Method for multiplexed nucleic acid patch polymerase chain reaction

ABSTRACT

The invention encompasses a method for amplifying at least two different nucleic acid sequences. In particular, the method encompasses a multiplexed nucleic acid patch polymerase chain reaction.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. provisional application No. 61/094,660, filed Sep. 5, 2008, which is hereby incorporated by reference in its entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under 5P50HG003170-0 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention encompasses a method for a amplifying at least two different nucleic acid sequences.

REFERENCE TO SEQUENCE LISTING

A paper copy of the sequence listing and a computer readable form of the same sequence listing are appended below and herein incorporated by reference. The information recorded in computer readable form is identical to the written sequence listing, according to 37 C.F.R. 1.821 (f).

BACKGROUND OF THE INVENTION

As the genes involved in various aspects of human physiology are elucidated, there are increasingly more candidate genes associated with disease. The application of this knowledge both in the clinic and to clinical research can be very powerful as the field moves toward personalized medicine. Examples of success include the sequencing of candidate disease loci in targeted populations, such as Ashkenazi Jews (Weinstein 2007), the sequencing of variants in drug metabolism genes to adjust dosage (Marsh and McLeod 2006), and the identification of genetic defects in cancer that make tumors more responsive to certain treatments (Marsh and McLeod 2006). However, the sequencing of many candidate genes across many individual samples necessitates the development of new technology to lower the cost and increase the throughput of medical re-sequencing to make clinical application more feasible.

The cost of sequencing is declining rapidly due to second generation sequencing technologies that perform a large number of sequencing reactions in parallel while using a small amount of reagent per reaction (Metzker 2005). These technologies integrate cloning and amplification into the sequencing protocol, which is essential for achieving the greater than 100-fold cost savings over traditional methods. However, this integration results in a loss of flexibility—it is not yet feasible to sequence a subset of the human genome in a large number of samples for the same cost as sequencing the complete genome of a single individual. This is a limitation, because sequencing the complete genome of a large numbers of individuals is still cost prohibitive, and the whole genome sequence of only a few individuals does not provide enough statistical power to make correlations between genotype and phenotype. The promise of personalized medicine based on genome analysis still glows on the horizon, but the significance behind observed variability is dim without an affordable technology to drive the necessary depth of patient sampling.

Current methods for analyzing sequence variation in a subset of the human genome rely on PCR to amplify the targeted sequences (Greenman et al. 2007; Sjoblom et al. 2006; Wood et al. 2007). Efforts to multiplex PCR have been hampered by the dramatic increase in the amplification of mispriming events as more primer pairs are used (Fan et al. 2006). In addition, large numbers of primer pairs often result in inter-primer interactions that prevent amplification (Han et al. 2006). Therefore, separate PCRs for each region of interest are performed, a costly approach when hundreds of individual PCRs must be performed for each sample (Greenman et al. 2007; Sjoblom et al. 2006; Wood et al. 2007). Furthermore, this strategy requires a large amount of starting DNA to supply enough template for all of the individual PCR reactions. This can be a problem as DNA is often a limiting factor when working with clinical samples.

It is important to choose the appropriate strategy for sample tracking to fully harness the throughput of second generation sequencing technologies. The sequencing capacities of these platforms are large enough that multiple samples can be sequenced with a single instrument run. To do this, one can use a separate compartment for each sample, but this only allows for a small number of samples, and there is a reduction in the total amount of sequence generated per run. Recently, Parameswaran et al. (Parameswaran et al. 2007) demonstrated the power of using DNA barcodes to label samples so that they can be pooled and sequenced together on the 454/Roche GS20 Sequencer. They were able to utilize the full capacity of the instrument and still determine from which sample each read originated. To realize the full power of second generation sequencing technologies, a multiplexing strategy should be compatible with DNA barcoding to track samples.

Therefore, there remains a need in the art for a multiplexed PCR method that simultaneously amplifies many targeted regions from a small amount of nucleic acid. The PCR method should also be compatible with next generation high throughput sequencing technologies where numerous samples can be processed in a single run. The PCR method should be specific and sensitive enough for identifying SNPs and mutations in individual samples.

SUMMARY OF THE INVENTION

One aspect of the present invention encompasses a method of amplifying at least two different nucleic acid sequences. Generally speaking, the method comprises the following steps: (a) defining the ends of at least two nucleic acid sequences; (b) annealing upstream and downstream nucleic acid patches to each nucleic acid sequence of step (a), and annealing an upstream universal primer to the upstream patch, and a downstream universal primer to the downstream patch; (c) ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence; and (d) amplifying the nucleic acid sequences of step (c).

Another aspect of the invention encompasses a method of amplifying one or more than one unique nucleic acid sequences. The method typically comprises the following steps: (a) annealing an upstream primer and a downstream primer to each unique nucleic acid sequence, wherein the upstream primer and the downstream primer comprise uracil instead of thymine; (b) amplifying each nucleic acid sequence so as to create amplicons of the nucleic acid sequence; (c) removing the upstream and downstream primer sequences from the amplicons of step (b) by contacting the amplicons with a uracil DNA glycosylase, an endonuclease, and an exonuclease; (d) annealing upstream and downstream nucleic acid patches to each unique amplicon of step (c), and annealing an upstream universal primer to the upstream patch of each unique amplicon, and a downstream universal primer to the downstream patch of each unique amplicon, wherein the downstream universal primer comprises a protecting group; (e) ligating the upstream universal primer and the downstream universal primer to each unique amplicon; (f) degrading non-specific amplicons of step (e); and (g) amplifying the amplicons of step (f).

Other aspects and iterations of the invention are described more thoroughly below.

REFERENCE TO COLOR FIGURES

The application file contains at least one photograph executed in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic of nucleic acid patch PCR. (A) A PCR reaction containing primers pairs for all targets is performed on genomic DNA. The primers contain uracil substituted for thymine. The primers are then cleaved from the amplicons by the addition of heat-labile Uracil DNA Glycosylase, Endonuclease VIII, and single strand specific Exonuclease I. (B) The ends of the target regions are now internal to the PCR primers (nested). (C) Nucleic acid patch oligonucleotides are annealed to the target amplicons and serve as a patch between the correct amplicons and universal primers. The universal primers are then ligated to the amplicons. The universal primer on the 3′ end of the amplicon is modified with a 3 carbon spacer that protects the selected amplicon from the final exonuclease reaction that degrades nonspecific products. (D) The selected amplicons are then amplified together simultaneously by PCR with universal primers.

FIG. 2 depicts a schematic of Sample Specific Barcode PCR. Sample-specific DNA barcodes are incorporated into the primers that are used for the final universal PCR. The 5′ end of the universal primer (white) is tailed with the sequences for the Roche/454 FLX Machine (grey) and sample-specific DNA sequences (black). When sequencing from either 454A or 454B, the first few bases indicate from which sample the read originated.

FIG. 3 shows the quantification of the abundance and reproducibility of nucleic acid patch PCR per exon in each sample. (A) Uniform Exon Abundance. Graph of the number of reads obtained for each targeted exon from the colon cancer sample and adjacent normal tissue. The 90 exons for which at least 1 read was obtained are ordered by abundance in the normal sample on the x-axis. The median number of reads per exon is 145. Seventy-six percent of all exons fell within 5 fold coverage of this median. All exons are within 3 log 10 of each other. (B) Correlation of number of reads across samples. Each exon is depicted as a point on the graph, where the x-axis is the number of reads in the normal sample and the y-axis is the number of reads in the colon cancer sample. The correlation was high (R² of 93%), indicating high reproducibility across samples. (C) Fold difference in abundance across samples. We computed the fold change of abundance per exon between the two samples. 85% (77/90) of exons displayed a 2 fold or less difference in abundance between samples. 100% of exons displayed a 3 fold or less difference in abundance between samples. Dotted line indicates 3 fold change.

FIG. 4 depicts a schematic of bisulfite nucleic acid patch PCR with ends defined by AluI digest. Genomic DNA is digested with AluI restriction enzyme. Nucleic acid patch oligonucleotides are then annealed to the target amplicons and serve as a patch between the correct amplicons and universal primers. The universal primers are then ligated to the amplicons. The universal primer on the 3′ end of the amplicon is modified with a 3 carbon spacer that protects the selected amplicon from the final exonuclease reaction that degrades nonspecific products. The reactions are then treated with sodium bisulfite to convert unmethylated cytosines to uracil. The selected amplicons are then amplified together simultaneously by PCR with universal primers.

FIG. 5 shows an image of the agarose gel electrophoresis of the final Universal PCR products of bisulfite nucleic acid patch PCR with ends defined by AluI digest. Each reaction was performed using decreasing quantities of starting human genomic DNA, as labeled in the figure. The expected smear of products is seen in the lanes that contained 900, 675, 450, 225, 112, 70, 50, and 20 ng of genomic DNA. The first lane contains Low Molecular Weight Ladder (NEB), with band sizes denoted on the left.

FIG. 6 depicts the sequencing results of bisulfite nucleic acid patch PCR with ends defined by AluI digest. The Y axis on the graph represents the number of reads obtained for each promoter. The promoters are order by length (bp) on the X axis.

FIG. 7 depicts a schematic of multiplexed bisulfite PCR. (A & B) Genomic DNA restriction digest. (C) Anneal patch oligos and universal primers specifically to the ends of desired fragments. (D) Ligate universal primers (U1 & U2) to targeted fragments. (E) Degrade unselected DNA with exonucleases. Targeted loci are protected from exonuclease by 3-prime modification on U2. (F) Treat with sodium bisulfite to convert unmethylated cytosine to uracil, leaving methylated cytosine intact. (G) PCR all loci simultaneously with universal primers tailed with sample-specific-DNA barcodes and sequencing machine primers (454A & 454B). Pool PCR products from all samples together for sequencing.

FIG. 8 depicts a photograph of an agarose gel showing that multiplexed bisulfite PCR works from small quantities of human genomic DNA. Image of the final universal PCR products by 3% Metaphor agarose gel electrophoresis. Each reaction was performed on a different amount of starting human genomic DNA, as labeled at the top of the figure. The expected smear of products is seen in the lanes that contained between 900 ng and 20 ng DNA. The gel image demonstrates that the reaction generates the expected products when as little as 20 ng of genomic DNA is used. A faint smear is visible in the lane that started with 1.6 ng in images taken at higher exposure.

FIG. 9 depicts two graphs showing the bisulfite method performance. (A) Number of sequencing reads per promoter for all 94 targeted promoters, order by length in base-pairs (bp) on the x-axis. Longer promoter amplicons yield fewer sequencing reads (length bias), but 87 amplicons (93%) have coverage within 10 fold of the median coverage (444 reads) The abundance of each promoter ranged from 10 to 5114 reads. (B) Histogram of the pair-wise squared correlation coefficients for the number of reads per promoter for all 48 samples. The mean correlation coefficient is 0.91, indicating that the number of reads per promoter is highly reproducible across patient samples.

FIG. 10 depicts an illustration of methylation at the H19 imprinted locus. Data from four patients who were germline heterozygous for a SNP (rs2251375) in this locus. The sequencing reads are aligned as rows in each panel. Each base in the read is color coded to indicate the sequence, yellow indicates a methylated cytosine, blue indicates all other bases. The position of the SNP is indicated by the red and white column, where a red base indicates reads from the G allele, and a white base indicates reads from the T allele. The percent of reads for each patient that are from the G allele is listed below the patient identifier for each sample. As expected for an imprinted locus, methylation is observed on one allele in both the tumor (left panels) and adjacent normal tissue (right panels) for each patient. Both alleles and both methylated and unmethylated molecules were amplified and sequenced efficiently from this locus in all samples.

FIG. 11 depicts an illustration of four promoters that exhibit tumor specific methylation. Sequencing reads from all patients for each type of tissue are grouped together in panels; breast tumors, adjacent normal breast tissues, colon tumors, and adjacent normal colon tissues. The sequencing reads are aligned as rows in each panel, and grouped by patient. Each base in the read is color coded to indicate the sequence, yellow indicates a methylated cytosine, blue indicates all other bases. (A & B) ICAM5 and LAMA1 promoters exhibit colon and breast tumor specific methylation. (C&D) KCNQ5 and CLSTN2 promoters exhibit colon tumor specific methylation.

FIG. 12 depicts an illustration of allelic tumor specific methylation. Data from six patients who are germline heterozygous for a SNP (rs2854744) in IGFBP3 promoter. The sequencing reads are aligned as rows in each panel. Each base in the read is color coded to indicate the sequence, yellow indicates a methylated cytosine, blue indicates all other bases. The position of the SNP is indicated by the red and white column, where red indicates reads from the A allele, and the C allele is indicated by yellow, if methylated, or white, if unmethylated and converted to a T. Patient ‘Breast 8’ is unmethylated on both alleles in both the tumor (left column) and normal tissue (right column). Patients ‘Breast 4’ and ‘Colon 6’ display tumor-specific methylation on only one allele, and the methylated allele differs between them. Patients ‘Colon 7’ and ‘Colon 12’ display tumor specific methylation on both alleles. Patient ‘Colon 12’ displays different patterns of methylation on each allele in the tumor.

FIG. 13 depicts a schematic of nucleic acid patch PCR with ends defined by oligo-directed FokI digestion. FokI-directing DNA oligonucleotides anneal upstream and downstream of target nucleic acid sequence in genomic DNA. These oligonucleotides contain a FokI restriction endonuclease recognition sequence, which directs FokI digestion of genomic DNA, defining the ends of the PCR template. Nucleic acid patch oligonucleotides are then annealed to the target amplicons and serve as a patch between the correct amplicons and universal primers. The universal primers are then ligated to the amplicons. The universal primer on the 3′ end of the amplicon is modified with a 3 carbon spacer that protects the selected amplicon from the final exonuclease reaction that degrades nonspecific products. The selected amplicons are then amplified together simultaneously by PCR with universal primers.

FIG. 14 shows an image of the agarose gel electrophoresis of the final Universal PCR products of nucleic acid patch PCR with ends defined by oligo-directed FokI digestion. The first lane contains Low Molecular Weight Ladder (NEB), with band sizes denoted on the left. The second lane contains the full reaction and a smear of products in the expected size range is achieved. The remaining lanes are each missing a component of the reaction, demonstrating that all components of the reactions (except Tween) are required to obtain the expected products.

FIG. 15 depicts schematic illustrations of various embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

PCR amplifies specific nucleic acid sequences through a series of manipulations including denaturation, annealing of oligonucleotide primer pairs, and extension of the primers with DNA polymerase. These steps can be repeated many times, potentially resulting in large amplification of the number of copies of the original target sequence. Multiplex PCR is a variant of PCR that enables the simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. However, current multiplex PCR methods are hampered by the amplification of mispriming events and inter-primer interactions that prevent amplification, as more primer pairs are used.

The present invention provides a method of multiplex PCR that affords a high level of specificity. The method also allows for parallel sequencing of multiple PCR amplification samples in a single sequencing run. Additionally, the invention provides uses for the method. Each is described in more detail below.

I. Nucleic Acid Patch PCR Method

Generally speaking, the method comprises defining the ends of at least two nucleic acid sequences, annealing upstream and downstream nucleic acid patches to each nucleic acid sequence, annealing an upstream and a downstream universal primer to each patch, and subsequently ligating the universal primers to each nucleic acid sequence. The resulting modified nucleic acid sequences may be amplified using primer sequences wherein each primer comprises a nucleic acid sequence tag specific for the sample, and a nucleic acid sequence to prime the sequencing reaction.

(a) Nucleic Acid Template

A method of the invention may be used to amplify nucleic acid sequences. Usually, the nucleic acid sequences may be found in a nucleic acid template. A nucleic acid template may be from any sample that contains nucleic acid molecules. The nucleic acid template may be from humans, animals, plants, microorganisms or viruses. In preferred embodiments, the nucleic acid template is from a human sample. The sample may be fresh, from archeological or forensic samples, or from preserved samples such as paraffin-embedded tissue. The sample may be a solid tissue or a physiological fluid, such as blood, serum, plasma, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, lymphatic fluid, mucous, synovial fluid, peritoneal fluid, or amniotic fluid. Nucleic acid templates may be prepared from the sample using methods well known to those of skill in the art (see, e.g., Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual,” 2^(nd) Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor). Alternatively, the sample containing the nucleic acid template may be used directly.

The nucleic acid template may be DNA, RNA, or a complementary DNA (cDNA) sequence that is synthesized from a mature messenger RNA. If the nucleic acid template is RNA, the RNA may be reverse transcribed to DNA using methods well known to persons skilled in the art. In a preferred embodiment, the nucleic acid template is DNA.

In some embodiments, suitable quantities of nucleic acid template for the invention may be 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 μg or less. In preferred embodiments, suitable quantities of nucleic acid template for the invention may be 1000, 900, 675, 450, 225, 112, 70, 50, 20, 1.6, 0.8, 0.4 ng or less.

In some embodiments, the nucleic acid template may be treated to prepare the template for specific applications of the invention. In one embodiment, the nucleic acid template may be treated with bisulfite to determine the pattern of methylation. Nucleic acid templates may be treated with bisulfite using methods well known to those of skill in the art, and may be performed using commercially available reagents, following manufacturer's protocols, such as by using the EZ DNA Methylation-Gold Kit™ (Zymo Research), the Imprint™ DNA Modification Kit (Sigma), or the like.

(b) Creation of Nucleic Acid Sequences with Defined Ends

The invention encompasses methods for the creation of nucleic acid sequences with defined ends. As used herein, the phrase “defined ends” refers to a nucleic acid sequence where both the 5′ and 3′ end of the sequence is known. Generally speaking, at least three, four, five, six, seven, or more than seven bases of the sequence are known. Non-limiting examples of methods for creating defined ends may include amplification (such as multiplex amplification), restriction endonuclease digestion, single strand specific exonuclease degradation, or triplex formation and cleavage. These methods are described in more detail below.

i. Multiplex Amplification from a Nucleic Acid Sample

Creating defined ends by multiplex amplification may consist of a PCR reaction using primer pairs for desired targets on the nucleic acid template. An exemplary example of a multiplex PCR reaction is depicted in FIG. 15A. Components of the multiplex PCR amplification reaction may include the nucleic acid sequence to be amplified (template; see section I(a) above), one or more primer pairs for delineating the target nucleic acid sequence on the template to be amplified (described below), one or more nucleotide polymerase (described below), deoxynucleotides, and salts and buffers essential for optimal activity of the polymerases in the reaction.

A. Primers

In a method for creating defined ends, the oligonucleotide PCR primers may be typically synthesized using the four naturally occurring deoxynucleotides dATP, dTTP, dCTP and dGTP. In some embodiments of this invention, oligonucleotide primers may also incorporate natural or synthetic deoxynucleotide analogs not normally present in DNA. Incorporation of nucleotide analogs, depicted as “x” in the diagram above, allows for the oligonucleotide primers to be selectively removed (see section (b) below) after amplification of the target nucleic acid. In some embodiments of the invention, a primer may be used such that, at one or more positions of the primer, one or more of the four deoxyribonucleotides in the primer may be replaced with one or more nucleotide analogs. Primers with nucleotide analogs located throughout the primer may also be used. In one preferred embodiment, primers may have one of the deoxynucleotides replaced with a nucleotide analog. In another preferred embodiment, 25%, 30%, 35% 40%, 50%, 60%, 70%, 80%, 90% or 100% of either dATP, dTTP, dCTP or dGTP in the primers may be replaced with a nucleotide analog. In yet another preferred embodiment, the nucleotide analog may be at the 3′-terminus of the primer.

PCR primers may be designed using standard primer design computer software techniques known to individuals skilled in the art. The variables considered during PCR primer design may include primer length, GC pair content, melting temperature, and size of the target nucleic acid amplified by the primer pair. Generally speaking, primers should not form hairpin structures or self- or hetero-primer pairs. In a preferred embodiment, primers may comprise a sequence of 15, 20, 25, 30, 35, 40, 45, 50 or more bases complementary to a portion of a template. In another preferred embodiment, the primer melting temperature may be 50, 55, 60, 65, 70 or 75° C. In a preferred embodiment, the primer melting temperature may be 61, 62, 63, 64, 65, 66 or 67° C. In one embodiment, the melting temperature of each primer of the primer pair may be the same. In another embodiment, the melting temperature of each primer of the primer pair may be different for each primer. In yet another embodiment, the difference in melting temperatures between each primer of the primer pair may be 1, 2, 3, 4, 5, 6, 7, 8, 9° C. or more. In another preferred embodiment, the maximum difference in melting temperature between primer pairs may be 5° C. In a preferred embodiment, the GC content of primer may be 10, 20, 30, 40, 50, 60, 70 or 80%. In yet another preferred embodiment the primer pair may be designed to amplify nucleic acid target products that may be 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or more base pairs in length.

B. Nucleotide Polymerases

In one embodiment of a method for creating defined ends, the nucleotide polymerase may be a DNA polymerase. In another embodiment, the nucleotide polymerase may be a thermostable polymerase. In a preferred embodiment, the nucleotide polymerase may be a thermostable DNA polymerase. A thermostable polymerase is an enzyme that is relatively stable to heat and eliminates the need to add enzyme prior to each PCR cycle. Non-limiting examples of thermostable polymerases may include polymerases isolated from the thermophilic bacteria Thermus aquaticus (Taq polymerase), Thermus thermophilus (Tth polymerase), Thermococcus litoralis (Tli or VENT™ polymerase), Pyrococcus furiosus (Pfu or DEEPVENT™ polymerase), Pyrococcus woosii (Pwo polymerase) and other Pyrococcus species, Bacillus stearothermophilus (Bst polymerase), Sulfolobus acidocaldarius (Sac polymerase), Thermoplasma acidophilum (Tac polymerase), Thermus rubber (Tru polymerase), Thermus brockianus (DYNAZYME™ polymerase) Thermotoga neapolitana (Tne polymerase), Thermotoga maritime (Tma) and other species of the Thermotoga genus (Tsp polymerase), and Methanobacterium thermoautotrophicum (Mth polymerase). The PCR reaction may contain more than one thermostable polymerase enzyme with complementary properties leading to more efficient amplification of target sequences. For example, a nucleotide polymerase with high processivity (the ability to copy large nucleotide segments) may be complemented with another nucleotide polymerase with proofreading capabilities (the ability to correct mistakes during elongation of target nucleic acid sequence), thus creating a PCR reaction that can copy a long target sequence with high fidelity. The thermostable polymerase may be used in its wild type form. Alternatively, the polymerase may be modified to contain a fragment of the enzyme or to contain a mutation that provides beneficial properties to facilitate the PCR reaction. In one embodiment, the thermostable polymerase may be Taq polymerase. Many variants of Taq polymerase with enhanced properties are known and include AmpliTaq™, AmpliTaq™ Stoffel fragment, SuperTaq™, SuperTaq™ plus, LA Taq™, LApro Taq™, and EX Taq™. In a preferred embodiment, the thermostable polymerase used in the multiplex amplification reaction of the invention is the AmpliTaq Stoffel fragment.

C. PCR Reaction Conditions

Buffer conditions for PCR reactions are known to those of ordinary skill in the art. PCR buffers may generally contain about 10-50 mM Tris-HCl pH 8.3, up to about 70 mM KCl, about 1.5 mM or higher MgCl₂, to about 50-200 μM each of dATP, dCTP, dGTP and dTTP, gelatin or BSA to about 100 μg/ml, and/or non-ionic detergents such as Tween-20 or Nonidet P-40 or Triton X-100 at about 0.05-0.10% v/v. In some embodiments, betaine may be added to the PCR reactions at about 0.25 to about 1 M. An example of a detailed description of buffer conditions may be found in Example 2.

In some embodiments, the multiplex PCR reaction may contain 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 or more primer pairs. Not all primer pairs will amplify targets with the same efficiency. In some embodiments, PCR primer pairs with similar amplification efficiency may be pooled in separate multiplex PCR reactions to have better representation of all targets. These PCR reactions may be combined after amplification.

In other embodiments, PCR amplification may be performed at a uniform temperature (isothermal PCR). Examples of isothermal PCR methods may include the ramification amplifying method and the helicase-dependent amplification method. In a preferred embodiment of the invention, PCR amplification may be by thermal cycling between a high temperature to melt the nucleic acid strands, a lower temperature to anneal the primers to the target nucleic acid, and an intermediate temperature compatible with the nucleic acid polymerase to elongate the nucleic acid sequence. In one embodiment, the melting temperatures may be about 85, 86, 87, 88, 89, 90, 95, or 100° C. In a preferred embodiment, the melting temperature may be about 90, 91, 92, 93, 94, 95, 96, 97 or 98° C. In another embodiment, the annealing temperatures may be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75° C. or more. In a preferred embodiment, the annealing temperature may be 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71, or 72° C. In yet another embodiment, the elongation temperature may be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80° C. or more. In a preferred embodiment, the elongation temperature may be 70, 71, 72, 73, 74, 75, 80° C. or more.

In certain embodiments, the PCR reaction may be incubated at the melting temperature for about 5 to about 60 seconds. In a preferred embodiment, the PCR reaction may be incubated at the melting temperature for about 30 seconds. In some embodiments, the PCR reaction may be incubated at the annealing temperature for about 5 to about 60 seconds. In a preferred embodiment, the PCR reaction may be incubated at the annealing temperature for about 30 seconds. In some embodiments, the PCR reaction may be incubated at the elongation temperature for about 1 to about 10 minutes. In a preferred embodiment, the PCR reaction may be incubated at the elongation temperature for about 6 minutes. In some embodiments, the PCR reaction is pre-incubated at the melting temperature for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes before cycling between the melting, annealing and elongation temperatures. In a preferred embodiment, the PCR reaction may be pre-incubated at the melting temperature for about 2 minutes.

In several embodiments, the PCR reactions may be cycled between the melting, annealing and elongation temperatures 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or more times. In a preferred embodiment, the PCR reactions may be cycled between the melting, annealing and elongation temperatures 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times.

D. Trimming Amplicons

In some embodiments of the invention, the amplified targets from the PCR reaction described above may be trimmed so the ends of the target regions become internal to the PCR primer sequences as depicted in FIG. 15B. The extent of the trimming may generally be defined by synthetic nucleotide analogs incorporated into the primer pairs described above. Treatments that specifically remove synthetic nucleotide analogs have been devised and are well known to those skilled in the art.

In certain embodiments, oligonucleotides containing 5-bromodeoxyuridine (BdUR) or 5-bromodeoxycytidine (BrdC) may be used as the primers of the invention. Primers containing BdUR may be degraded upon exposure to light. In other embodiments, the deoxyinosine may be incorporated into primers of the invention. Primers containing deoxyinosine may be degraded using Endonuclease V, an enzyme that recognizes and cleaves the sugar phosphate backbone at the deoxyinosine residue.

In other embodiments, the base of the synthetic nucleotide is first specifically removed, leaving an apurinic or apyrimidinic site (AP site) and an intact sugar-phosphate backbone. The sugar-phosphate backbone is then cleaved at the AP site, generating a nick in the target, which dictates the nucleic acid sequence to be removed by exonuclease enzymes. In preferred embodiments of the invention, the base of the synthetic nucleotide analog is removed with a DNA glycosylase enzyme. DNA glycosylases are a family of enzymes that can remove the base of some nucleotide analogs. Some examples of nucleotide analogs that may be incorporated into primers and that are substrates for glycosylase enzymes may include deoxyuridine, deoxy-7-methylguanosine, deoxy-5,6-dihydroxythymidine, deoxy-3-methyladenosine, deoxyinosine, 5-methyl-deoxycytidine, O-6-methyl-deoxyguanosine, 5-iodo-deoxyuridine, 8-oxy-deoxyguanine, and 1,N⁶-ethenoadenine. Glycosylase enzymes that remove bases from nucleotide analogs incorporated into target nucleic acid sequences may include uracyl DNA glycosylase, 7-methylguanine-DNA glycosylase, 5,6-dihydroxythymidine glycosylase, 3-methyladenine glycosylase, hypoxanthine DNA N-glycosylases, 8-oxoguanine-DNA glycosylase, and alkylpurine-DNA-N-glycosylase. In a preferred embodiment, the nucleotide analog may be deoxyuridine. In another preferred embodiment, the DNA glycosylase enzyme may be uracil DNA glycosylase.

In some embodiments, treatments that cleave AP sites may include, but are not limited to, heat, alkaline hydrolysis, tripeptides such as Lys-Trp-Lys and Lys-Tyr-Lys, AP endonucleases such as endonuclease III, endonuclease IV, endonuclease VI, endonuclease VIII, phage T4 UV endonuclease, and the like. In a preferred embodiment, the treatment is endonuclease VIII.

After removing primers from amplified target nucleic acid sequences, the resulting single strand overhanging nucleic acid sequence at the 3′ termini may be removed using an enzyme with a 3′ to 5′ single stranded exonuclease activity as depicted in the diagram above. Commonly used 3′ to 5′ exonucleases that remove single stranded nucleic acids may include exonuclease I and exonuclease VII. In a preferred embodiment of the invention, the exonuclease is exonuclease I.

After trimming the ends of the amplified target nucleic acids, other manipulations that prepare the reactions for subsequent steps may be performed. For example, removal of unincorporated nucleotides might be required. In some embodiments, this may be accomplished by physical means such as precipitation, filtration, and chromatography. In other embodiments, the unincorporated nucleotides may be diluted to a concentration where they would not interfere in later steps. In preferred embodiments, the unincorporated nucleotides may be removed using enzymes such as apyrase, an ATP diphosphohydrolase that catalyses the removal of the gamma phosphate from ATP and the beta phosphate from ADP.

ii. Restriction Endonuclease Enzymes

In another embodiment, restriction endonuclease enzymes may be used to create nucleic acid sequences with defined ends. Suitable restriction endonuclease enzymes may include type I, type II, type III or type IV restriction endonuclease enzymes. Generally speaking, the restriction enzyme used should have recognition sites that flank, and not bisect, the desired nucleic acid sequence. In some embodiments, the restriction endonuclease enzymes may be type I restriction endonuclease enzymes. Non-limiting examples of Type I restriction endonuclease enzymes may include Cfrl, Eco377I, EcoAI, EcoDXXI, EcoKI, Eco124I, KpnAI, and StySPI. In other embodiments, the restriction endonuclease enzymes may be type II restriction endonuclease enzymes. Type II restriction endonuclease enzymes suitable for the methods of the invention may be a restriction endonuclease enzyme of type IIB, type IIE, type IIF, type IIG, type IIM, type IIS, or type IIT. In certain embodiments, Type III restriction endonuclease enzymes may be suitable for the methods of the invention. Non-limiting examples of Type III restriction endonuclease enzymes are known in the art. In alternative embodiments, the restriction endonuclease enzymes may be Type IIS restriction endonuclease enzymes. Non-limiting examples of Type IIS restriction endonuclease enzymes may include FokI, HgaI, EciI, BceAI, BbvI, BtgZI, BsmFI, BpmI, and BsgI. Other restriction endonuclease enzymes are known in the art. For instance, additional non-limiting examples may be found at http://rebase.neb.com/cgi-bin/azlist?re1, http://rebase.neb.com/cgi-bin/azlist?re2, http://rebase.neb.com/cgi-bin/azlist?re3, or http://rebase.neb.com/cgi-bin/azlist?re4.

The restriction endonuclease enzyme cut sites may be used to define the ends of nucleic acid templates. An exemplary example of a restriction enzyme reaction creating nucleic acid sequences with defined ends is depicted in FIG. 15C. Components of the restriction enzyme reaction may include the nucleic acid sequence to be digested (template; see section I(a) above), one or more restriction endonucleases, and salts and buffers essential for optimal activity of the enzymes in the reaction. The restriction enzyme reaction may be prepared using methods well known to those of skill in the art (see, e.g., Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual,” 2^(nd) Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor).

In some embodiments, oligonucleotides may be used to direct Type IIs restriction enzymes to cut at specific sites in the nucleic acid template. As depicted in FIG. 15D, this is facilitated by upstream and downstream oligonucleotides that anneal upstream and downstream of the target nucleic acid sequences and serve as a guide for digestion by the type IIs restriction endonuclease enzyme. Thus, components of the restriction enzyme reaction may include the nucleic acid sequence to be digested (template; see section I(a) above), one or more restriction endonucleases, the oligonucleotides directing the restriction endonuclease cut sites (described below), and salts and buffers essential for optimal activity of the enzymes in the reaction.

A. Oligonucleotides Directing Type IIs Restriction Endonuclease Enzymes

The upstream and downstream restriction enzyme-directing oligonucleotides may be designed using primer length, GC pair content, and melting temperature criteria as described in section I(b)iA above. In some preferred embodiments, the 5′ ends of the upstream restriction enzyme-directing oligonucleotides may be complementary to a portion of the desired nucleic acid sequence (e.g. the segment parallel to the genomic DNA in the diagram above), and may be concatenated at the 3′ end of the oligonucleotides to double-stranded nucleotide sequences encoding type IIs restriction enzymes. In other preferred embodiments, the 3′ ends of the downstream restriction-enzyme-directing oligonucleotides may be complementary to a portion of the desired nucleic acid sequences (e.g. the segment parallel to the genomic DNA in the diagram above), and may be concatenated at the 5′ end of the oligonucleotides to double-stranded nucleotide sequences encoding type IIs restriction enzymes.

B. Annealing and Digestion Reaction Conditions

Annealing of the restriction enzyme-directing oligonucleotides to the nucleic acid templates may generally be performed before addition of the restriction enzyme for digestion. In addition to the nucleic acid template, annealing reactions may generally contain about 1 pM to about 500 nM of each restriction enzyme-directing oligonucleotide, and about 0.01 to about 0.9% Tween80. An example of a detailed description of buffer conditions may be found in Example 8.

In some embodiments, annealing of the restriction enzyme-directing oligonucleotides may be performed by melting the nucleic acid strands at a high temperature, followed by a lower temperature suitable for annealing the restriction enzyme-directing oligonucleotides to target nucleic acid sequences. In one embodiment, the melting temperatures may be about 85, 86, 87, 88, 89, 90, 95, or 100° C. In a preferred embodiment, the melting temperature may be about 90, 91, 92, 93, 94, 95, 96, 97 or 98° C. In another embodiment, the annealing temperatures may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55° C. or more. In a preferred embodiment, the annealing temperatures may be about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 50, 51, or 52° C.

In other embodiments, the annealing reactions may be incubated at the melting temperature for about 5 to about 30 minutes. In a preferred embodiment, the annealing reactions may be incubated at the melting temperature for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 minutes. In some embodiments, the annealing reactions may be incubated at the annealing temperature for about 1 to about 10 minutes. In a preferred embodiment, the annealing reactions may be incubated at the melting temperature for about 1, 2, 3, 13, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes.

After annealing the restriction enzyme-directing oligonucleotides to the template, the type IIs restriction enzyme may be added, and the restriction enzyme reaction may be prepared using methods well known to those of skill in the art (see, e.g., Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual,” 2^(nd) Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor).

iii. Single Strand Specific Exonuclease Degradation

Single strand specific exonuclease enzyme digestion of nucleic acid templates protected by locus-specific oligonucleotides may be used to define ends of the nucleic acid template. As depicted in FIG. 15E, this is facilitated by upstream and downstream oligonucleotides that anneal upstream and downstream of the target nucleic acid sequences and serve as protection against digestion by the single strand specific exonuclease enzymes. Thus, components of the exonuclease reaction may include the nucleic acid sequence to be digested (template; see section I(a) above), one or more single strand specific exonuclease enzymes (described below), the oligonucleotides protecting the nucleic acid template (described below), and salts and buffers essential for optimal activity of the exonucleases in the reaction.

Non-limiting examples of single strand specific exonuclease enzymes suitable for the methods of the invention may be exonuclease VII, exonuclease I, RecJ exonuclease, or Terminator™ 5′-Phosphate-Dependent Exonuclease (Epicentre Biotechnologies). The upstream and downstream oligonucleotides may be designed using primer length, GC pair content, and melting temperature criteria as described in (b)i.A. above.

Annealing of the protecting oligonucleotides to the nucleic acid templates may generally be performed before addition of the exonuclease enzymes. In addition to the nucleic acid template, annealing reactions may generally contain about 1 pM to about 500 nM of each oligonucleotide. In some embodiments, annealing of the oligonucleotides may be performed by melting the nucleic acid strands at a high temperature, followed by a lower temperature suitable for annealing the protecting oligonucleotides to target loci. In one embodiment, the melting temperatures may be about 85, 86, 87, 88, 89, 90, 95, or 100° C. In a preferred embodiment, the melting temperature may be about 90, 91, 92, 93, 94, 95, 96, 97 or 98° C. In another embodiment, the annealing temperatures may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55° C. or more. In a preferred embodiment, the annealing temperatures may be about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 50, 51, or 52° C.

In some embodiments, the annealing reactions may be incubated at the melting temperature for about 5 to about 30 minutes. In a preferred embodiment, the annealing reactions may be incubated at the melting temperature for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 minutes. In some embodiments, the annealing reactions may be incubated at the annealing temperature for about 1 to about 10 minutes. In a preferred embodiment, the annealing reactions may be incubated at the melting temperature for about 1, 2, 3, 13, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes. After annealing of the protecting oligonucleotides, the exonuclease enzymes may be added for digestion.

iv. Triplex Formation and Cleavage by Endonucleases

The ability of some nucleic acid recombination proteins to direct the formation of triplex nucleic acid structures may be used to create defined ends of a nucleic acid sequence. Triplex DNA structures are induced at specific loci by incubating nucleic acid templates with locus-specific oligonucleotides that have been coated with the recombination protein. The triplex structure then produces a single stranded region of nucleic acid available for cleavage by single strand specific endonucleases. Thus, components of the restriction enzyme reaction may include the nucleic acid sequence to be digested (template; see section I(a) above), one or more recombination proteins, the recombination protein-coated locus-specific oligonucleotides, the endonuclease proteins, and salts and buffers for optimal activity of the enzymes. Non-limiting examples of recombination proteins may include RecA of Escherichia coli, or any homologous recombination protein capable of inducing formation of triplex DNA structure. Non-limiting examples of single strand specific endonucleases may include S1 and BAL1 endonucleases.

(c) Nucleic Acid Patch PCR

One aspect of the invention is the ligation of universal primer sequences to nucleic acid sequences. As depicted in FIG. 15F, this is facilitated by upstream and downstream nucleic acid patch oligonucleotides that anneal upstream and downstream of the target nucleic acid sequences and serve as a patch between the desired sequence and upstream and downstream universal primers to be ligated. Thus, nucleic acid patch ligation reactions contain the target sequences, the upstream and downstream universal primers to be ligated, the upstream and downstream nucleic acid patch oligonucleotides to guide the specific ligation of the universal primers, and the enzymes and other components needed for the ligation reaction. In preferred embodiments, target sequences may be nucleic acid sequences with defined ends as described above.

i. Universal Primers

The upstream and downstream universal primers may be designed using primer length, GC pair content and melting temperature criteria as described in I(a) above. In some embodiments, the downstream universal primer may be modified to facilitate further steps of the invention. In a specific embodiment, the downstream universal primer may be modified with a 5′ phosphate group to enable ligation of the downstream universal primer to the amplicon. In other specific embodiments, the 3′ end of the downstream universal primer may be modified for protection against exonuclease digestion. Modifications at the 3′ end may be introduced at the time of synthesis or after synthesis through chemical means well know to those of skill in the art. Modifications may be 3′ terminal or slightly internal to the 3′ end. Some examples of modifications that make nucleic acid sequences exonuclease resistant include, but are not limited to, locked nucleic acids (LNA's), 3′-linked amino groups, 3′ phosphorylation, the use of a 3′-terminal cap (e.g., 3′-aminopropyl modification or by using a 3′-3′ terminal linkage), phosphorothioate modifications, the use of attachment chemistry or linker modification such as Digoxigenin NHS Ester, Cholesteryl-TEG, biotinylation, thiol modifications, or addition of various fluorescent dyes and spacers such as C3 spacer. In a preferred embodiment, the downstream universal primer is protected from exonuclease digestion by a C3 spacer.

ii. Nucleic Acid Patch Primers

In some embodiments, an upstream and a downstream nucleic acid patch oligonucleotide may be designed for each amplicon (see diagram above). In some preferred embodiments, the 5′ ends of the upstream nucleic acid patch oligonucleotides may be complementary to sequences in the amplicons (grey segment of upstream nucleic acid patch oligonucleotide in diagram above), and may be concatenated to upstream nucleotide sequences complementary to the upstream universal primer sequence on the 3′ end (black segment of upstream nucleic acid patch oligonucleotide in diagram above). In other preferred embodiments, the 3′ ends of the downstream nucleic acid patch oligonucleotides may be complementary to downstream sequences in the amplicons (grey segment of downstream Nucleic acid patch oligonucleotide in diagram above), and may be concatenated to nucleotide sequences complementary to the downstream universal primer sequence on the 5′ end (black segment of downstream nucleic acid patch oligonucleotide in diagram above).

iii. Ligation of Universal Primers

In some embodiments, the universal primers may be ligated to nucleic acid sequences. In a process similar to a PCR amplification reaction, multiple cycles of heating and cooling may be used to melt the target nucleic acid sequence, anneal the nucleic acid patch and universal primers, and ligate the universal primers to target nucleic acid sequences.

In some embodiments of the invention, the universal primers of the invention may be ligated to the target nucleic acids using a DNA ligase. The ligase may be thermostable. In preferred embodiments, the ligase is a thermostable DNA ligase. A thermostable DNA ligase is an enzyme that is relatively stable to heat and eliminates the need to add enzyme prior to each ligation cycle. Non-limiting examples of thermostable DNA ligases may include Ampligase® Thermostable DNA Ligase, Taq DNA Ligase from Thermus aquaticus, Tfi DNA ligase from Thermus filiformis, Tth DNA ligase from Thermus thermophilus, Thermo DNA ligase, Pfu DNA ligase from Pyrococcus furiosus, and thermostable DNA ligase from Aquifex pyrophilus. The thermostable polymerase may be used in its wild type form, modified to contain a fragment of the enzyme, or to contain a mutation that provides beneficial properties to facilitate the ligation reaction. In a preferred embodiment, the thermostable ligase is Ampligase®.

iv. Ligation Reaction Conditions

Ligation reactions may generally contain about 1 pM to about 500 nM of each nucleic acid patch oligo, about 1 pM to about 500 nM of each universal primer, about 3, 4, 5, 6, 7, or 8 units of Ampligase®, and 1× Ampligase Reaction Buffer. An example of a detailed description of buffer conditions may be found in Example 2.

In some embodiments, ligation reactions may be performed by thermal cycling between a high temperature to melt the nucleic acid strands, a sequence of 1, 2, 3, 4 or 5 lower temperatures to anneal the nucleic acid patch oligonucleotides to the target nucleic acid, and a temperature compatible with the ligase to ligate the nucleic acid sequence. In a preferred embodiment, ligation reactions may be performed by thermal cycling between a high temperature to melt the nucleic acid strands, a first lower temperature to anneal the nucleic acid patch oligonucleotides to the target nucleic acid, a second lower temperature to anneal the universal primers to the nucleic acid patch oligonucleotides, and a temperature compatible with the ligase to ligate the nucleic acid sequence. In one embodiment, the melting temperatures may be about 85, 86, 87, 88, 89, 90, 95, or 100° C. In a preferred embodiment, the melting temperature may be about 90, 91, 92, 93, 94, 95, 96, 97 or 98° C. In another embodiment, the Nucleic acid patch oligonucleotide annealing temperatures may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75° C. or more. In a preferred embodiment, the nucleic acid patch oligonucleotide annealing temperatures may be about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71, or 72° C. In another embodiment, the ligation temperature may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80° C. or more. In a preferred embodiment, the ligation temperature may be about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70° C. or more.

In some embodiments, the ligation reactions may be incubated at the melting temperature for about 5 to about 60 seconds. In a preferred embodiment, the ligation reactions may be incubated at the melting temperature for about 30 seconds. In some embodiments, the ligation reactions may be incubated at the nucleic acid patch oligonucleotide annealing temperature for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more minutes. In a preferred embodiment, the reactions may be incubated at the nucleic acid patch oligonucleotide annealing temperature for about 2 minutes. In some embodiments, the ligation reactions may be incubated at the universal primer annealing temperature for about 30 seconds to about 5 minutes. In a preferred embodiment, the ligation reactions may be incubated at the universal primer annealing temperature for about 1 minute. In some embodiments, the ligation reactions may be incubated at the ligation temperature for about 30 seconds to about 5 minutes. In a preferred embodiment, the ligation reactions may be incubated at the ligation temperature for about 1 minute. In some embodiments, the reactions may be pre-incubated at the melting temperature for about 5, 6, 7, 8, 9, 10, 15, 20 or 25 minutes before cycling between the melting, annealing and ligation temperatures. In a preferred embodiment, the ligation reactions may be pre-incubated at the melting temperature for about 15 minutes.

In some embodiments, the ligation reactions may be cycled between the melting, annealing and ligation temperatures about 10, 50, 100, 150, 200 or more times. In a preferred embodiment, the ligation reactions may be cycled between the melting, annealing and elongation temperatures about 100 times.

(d) Degrade Mispriming Products and Genomic DNA

In some embodiments, exonucleases may be added to the ligation reaction at the completion of the reaction to degrade mispriming products of the multiplex PCR reaction or genomic DNA. In preferred embodiments, exonucleases may be 3′ to 5′ exonucleases. Exonucleases may be single stranded or double stranded exonucleases. Non-limiting examples of exonucleases suitable for this step of the reaction may include exonuclease I, exonuclease III and mung bean nuclease. One or more exonucleases may be added. In a preferred embodiment, the exonucleases may be exonuclease I and III.

(e) Sample-Specific Barcode PCR and Sequencing of Nucleic Acid Patch Amplicons

In some aspects of the invention, nucleic acid samples may be sequenced. In some embodiments, the nucleic acids sequenced may be the amplicons prepared in (a), (b) and (c) above. Sequencing techniques suitable for the invention may be high throughput. High throughput sequencing techniques may include techniques based on chain termination, pyrosequencing (sequence by synthesis), or sequencing by ligation and are well known to those of skill in the art. In some embodiments, high throughput sequencing techniques like true single molecule sequencing (tSMS) may not require amplification of target nucleotide sequences. In preferred embodiments, sequencing may be performed using high throughput sequencing techniques that involve in vitro clonal amplification of the target nucleotide sequence. Non-limiting examples of high throughput sequencing techniques that involve amplification may include solid-phase PCR in polyacrylamide gels, emulsion PCR, rolling-circle amplification, bridge PCR, BEAMing (beads, emulsions, amplification and magnetics)-based cloning on beads, massively parallel signature sequencing (MPSS) to generate clonal bead arrays. In a preferred embodiment, the amplicons may be sequenced using PCR techniques as exemplified by 454 Sequencing™. The PCR amplification for 454 sequencing may be as depicted in FIG. 15G.

In some embodiments, the PCR may use primers complementary to the universal primer sequences described in section I(c)i above, and depicted as black segments in the diagram. In other embodiments, the PCR primers may be coupled to nucleic acid sequences for sequencing (grey segments of the primers in diagram above). In a preferred embodiment, the primers for the final universal PCR may be tailed to 454 sequencing primers A and B (454 Life Sciences, Branford, Conn.). In other embodiments, the primers for the PCR amplification may be complementary to the upstream and downstream universal primer nucleotide sequences ligated in FIG. 15G (black segments of the primers). In additional embodiments, the PCR primers may be coupled to nucleic acid sequence barcodes (white segments of the primers in FIG. 15G). In some embodiments, the nucleic acid barcode may be about 4, 5, 6, 7, 8, 9, 10, or more bases. In a preferred embodiment, the nucleic acid barcode may be about 6 bases. The barcodes may be at the 5′ end, the 3′ end or, as exemplified in FIG. 15G, internal to the primer sequence.

In some embodiments, nucleic acid sequences amplified in the PCR reactions of more than one sample may be pooled for parallel sequencing of nucleic acids prepared in multiple samples. In some embodiments, about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or more samples may be pooled for sequencing.

II. Methods of Use

A further aspect of the invention provides uses for the amplification method detailed herein. In some embodiments, a method described herein may be used to detect and discover single nucleotide polymorphisms (SNPs) or mutations. In other embodiments, a method described herein may be used to detect pathogen DNA in a high background of host DNA, detect rare DNA to allow for multiplexed or genome-wide amplification of biomarkers in peripheral samples, or amplify targets from degraded samples to allow for multiplexed or genome-wide amplification. In a specific embodiment, a method described herein may be used to detect rare tumor DNA to allow for multiplexed or genome-wide amplification of biomarkers in peripheral samples such as blood or stool. In yet other embodiments, a PCR method described herein may be used to detect DNA methylation. Other applications that rely heavily on PCR may benefit from higher levels of multiplexing, such as the amplification of all exons or all conserved regions, or the engineered assembly of many DNA fragments simultaneously in synthetic biology experiments.

In still other embodiments, the PCR method described herein may be used to detect DNA methylation, detect and/or sequence tumor DNA derived from peripheral samples (blood, stool), amplify all exons in a particular template, or amplify all conserved regions in a particular template. A skilled researcher in the art will appreciate that other methods of use for a method detailed herein may be possible or desirable, and that the methods of use detailed herein are not to be construed as limiting.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

EXAMPLES

The following examples illustrate various iterations of the invention.

Example 1 Nucleic Acid Patch PCR Design

Mispriming events plague standard multiplex PCR reactions as the number of primer pairs increases. Nucleic acid patch PCR was designed to significantly decrease mispriming events, as nucleic acid patch PCR requires four oligonucleotide hybridizations per locus. This results in a more specific amplification than standard multiplex PCR, which requires only two hybridizations per locus. FIG. 1 presents a schematic of the concept of nucleic acid patch PCR.

In the first round of oligonucleotide hybridization, a PCR reaction containing DNA primer pairs for all targets is performed on genomic DNA (FIG. 1A). These DNA primers contain uracil substituted for thymine to facilitate the next step of the process. The PCR is performed for a low number of cycles and serves to define the ends of the target regions. To prepare for the second round of oligonucleotide hybridization, the PCR product generated above is first trimmed to produce a nucleic acid fragment with ends internal to the PCR primer sequences (FIG. 1B). This is accomplished by removing the uracil-containing primers, and trimming the resulting DNA overhangs on the PCR product by an enzyme mix containing uracil DNA glycosylase.

Next, a second round of oligonucleotide hybridization is performed. Nucleic acid patch oligonucleotides are annealed to the target amplicons and serve as a patch between the correct amplicons and universal primers (FIG. 1C). In the third oligonucleotide hybridization, the universal primers are annealed to the nucleic acid patch primers, and then ligated to the amplicons in a reaction containing a thermostable ligase followed by exonucleases I and III. This reaction provides two levels of selection in addition to the oligonucleotide hybridization. First, the thermostable ligase used is sensitive to mismatched bases near the ligation junction (Barany 1991), and second, the exonucleases in the reaction provide an added level of selectively by degrading mispriming products and the genomic DNA. The selected amplicons are protected from the exonuclease in the final reaction by a 3′ modification with a 3-carbon spacer on the universal primer. The selected amplicons are then amplified together simultaneously by PCR with the universal primers (FIG. 1D) for the final round of selection.

The target selection protocol is an addition-only reaction and can be performed in a single tube per sample, making it amenable to automation. To pool and sequence multiple samples, nucleic acid patch PCR is first performed separately for each sample (1 tube per sample). Sample-specific DNA barcodes are then incorporated into the primers used for the final universal PCR by tailing the 5′ end with sample-specific DNA sequences and 454 sequencing primers (FIG. 2). Thus, the first few bases indicate from which sample each read originated.

Example 2 Nucleic Acid Patch PCR and Sequencing of Candidate Genes in Colon Cancer

To demonstrate the multiplexed selection and amplification of exons by nucleic acid patch PCR described in Example 1, single nucleotide polymorphisms (SNPs) and mutations were analyzed in six nucleotide sequences encoding cancer related proteins: tumor protein p53 (TP53); adenomatous polyposis coli (APC); mutL homolog 1, colon cancer, nonpolyposis type 2 (MLH1); retinoblastoma 1 (RB1); breast cancer 1, early onset protein (BRCA1); and von Hippel-Lindau tumor suppressor protein (VHL) (Marsh and Zori 2002). These targets are located across 4 chromosomes, vary in length from 74 bp to 438 bp, and total 21.6 kbp. Oligonucleotide design, conditions of PCR reactions, sequencing and sequence analysis are described below.

Oligonucleotide Design

Human exon sequence plus 150 bp flanking sequence from the March 2006 assembly was downloaded from the UCSC Genome Browser (www.genome.ucsc.edu). The reference sequences (Refseq) representing the six colon cancer related nucleic acids were: NM_(—)000038 (APC), NM_(—)000546 (TP53), NM_(—)000249 (MLH1), NM_(—)000321 (RB1), NM_(—)007304 (BRCA1), and NM_(—)000551 (VHL). The convention that exon numbering for each gene begins with zero was maintained throughout the analysis. Primer3 software (http://frodo.wi.mit.edu/) was then used to select primer pairs flanking the exon. The design was constrained to PCR products between 50-500 bp, primer length 20-36 bp, primer melting temperature (Tm)=61-67° C., where the maximum difference in Tm between primer pairs was 5° C., and the GC content of the primer had to be between 10-80%. Four thousand possible primer pairs were generated per exon. Those primer pairs that ended with a T as the 3′ base were then selected. Oligonucleotide sequences of the PCR primers are listed in TABLE A. All PCR primer oligonucleotides were synthesized by Sigma-Genosys.

A nucleic acid patch oligonucleotide was then designed by extending into the sequence from the PCR primer until the Tm of the nucleic acid patch oligonucleotide was 62-67° C. The selected oligonucleotides were then aligned against themselves using BLASTN software from the Washington University BLAST Archives WUBLAST (http://blast.wustl.edu) to approximate cross reactivity. For each exon, the oligonucleotide sets with the fewest blastn matches to the entire set were chosen. The PCR primer sequence was substituted with a deoxyuridine in place of every deoxythymidine. The nucleic acid patch oligonucleotides were then concatenated with the complement universal primer sequences to result in the appropriate patch sequence. Sequences of the nucleic acid patch oligonucleotides are listed in TABLE B. All nucleic acid patch oligonucleotides were synthesized by Sigma-Genosys.

Two Universal Primer oligonucleotides were synthesized for the ligation reaction, including the Universal Primer 2, which has a 5′ Phosphate and a 3-carbon spacer on the 3′ end. The Universal Primer oligonucleotide sequences were then tailed at the 5′ end with the sample-specific DNA barcodes and 454 Life Sciences A or B oligonucleotide sequence to result in the Final Universal Primer oligonucleotides for normal samples and colon cancer samples. The Universal Primer oligonucleotides for ligation and the Final Universal primer oligonucleotide sequences for normal and colon cancer samples are listed in TABLE C.

TABLE A Multiplex PCR SEQ ID. NO. Oligo Name Sequence 1 000038_00_PCRleft TCTTAAGAGTTTTGTTTCCTTTACCCCU 2 000038_01_PCRleft CGTGCTTTGAGAGTGATCTGAATTU 3 000038_02_PCRleft TTGTGGTTAAAATGTAAACCTAATATTTCACU 4 000038_03_PCRleft GGTAGAGAAGTTTGCAATAACAACTGAU 5 000038_04_PCRleft AAATAATTTTCTCATGCACCATGACU 6 000038_05_PCRleft TTAAATGAGAATGATTTGACATAACCCU 7 000038_06_PCRleft AAAAAGCCTTGGGCTAAGAAAGCCU 8 000038_07_PCRleft AATGGTCATACTTTTATGATGTATTTAATTGTTU 9 000038_08_PCRleft GCTTTTGGATATTAAAGTCGTAATTTTGTTU 10 000038_09_PCRleft ATTTGTTGATCCACTAAAATTCCGU 11 000038_10_PCRleft TGATTGTCTTTTTCCTCTTGCCCTU 12 000038_11_PCRleft AAAGCTTGGCTTCAAGTTGTCTTTU 13 000038_12_PCRleft AAAGTGATAGGATTACAGGCGTGAGU 14 000038_13_PCRleft GAAGTTAATGAGAGACAAATTCCAACTCU 15 000249_00_PCRleft CCGTTGAGCATCTAGACGTTTCCU 16 000249_01_PCRleft CCTGTAAGACAAAGGAAAAACACGTTAAU 17 000249_02_PCRleft TGGATTAAATCAAGAAAATGGGAAU 18 000249_03_PCRleft CAGCAGTTCAGATAACCTTTCCCTTU 19 000249_04_PCRleft TGTTGATATGATTTTCTCTTTTCCCCTU 20 000249_05_PCRleft TGGATTCACTATCTTAAGACCTCGCTTU 21 000249_06_PCRleft GGGCTCTGACATCTAGTGTGTGTTU 22 000249_07_PCRleft TCCTTGTGTCTTCTGCTGTTTGTTU 23 000249_08_PCRleft GAGGACCTCAAATGGACCAAGTCU 24 000249_09_PCRleft GGTGATTTCATGACTTTGTGTGAATGU 25 000249_10_PCRleft ATCTTCTGGCCACCACATACACCAU 26 000249_11_PCRleft GCTCCATTTGGGGACCTGTATATCU 27 000249_12_PCRleft GCTCTGTAGAACCAGCACAGAGAAGTU 28 000249_13_PCRleft AGGCTTCTTTGCTTACTTGGTGTCU 29 000249_14_PCRleft TCTCATCCATGTTTCAGGGATTACU 30 000249_15_PCRleft TTGCTCCTTCATGTTCTTGCTTCTU 31 000249_16_PCRleft ATCAAGTAACGTGGTCACCCAGAGU 32 000249_17_PCRleft CAGCAATATTCAGCAGTCCCATTU 33 000249_18_PCRleft ATCAGCCAGGACACCAGTGTATGTU 34 000321_00_PCRleft GAAGTGACGTTTTCCCGCGGU 35 000321_01_PCRleft GATCTTAAAGTATTTAATAATGTTCTTTTTCACAGU 36 000321_02_PCRleft CCATCAGAAGGATGTGTTACAAATATACAGU 37 000321_03_PCRleft AATTCCTTCCAAAGGATATAGTAGTGATTU 38 000321_04_PCRleft TCTTAAAAGAAGATAAATAAAGCATGAGAAAACU 39 000321_05_PCRleft GCACAAAAAGAAACACCCAAAAGAU 40 000321_06_PCRleft CATGCTGATAGTGATTGTTGAATGAAU 41 000321_07_PCRleft GGATGTACAATTGTTCTTATCTAATTTACCACTU 42 000321_08_PCRleft CATGGGGGATTGACACCTCTAACU 43 000321_09_PCRleft AAAATTCTTTAATGAAATCTGTGCCTCU 44 000321_10_PCRleft TTATATGATTTTATGAGACAACAGAAGCATU 45 000321_11_PCRleft AACCACAGTCTTATTTGAGGGAATGU 46 000321_12_PCRleft CGACATTGATTTCTGTTTTTACCTCCU 47 000321_14_PCRleft TGAGCCAAGATTGTGCCATU 48 000321_15_PCRleft AATTATCTGTTTCAGGAAGAAGAACGAU 49 000321_16_PCRleft TGGTTTAACCTTTCTACTGTTTTCTTTGTCU 50 000321_17_PCRleft TTCATTCTGACTTTTAAATTGCCACU 51 000321_18_PCRleft TCTGGGTGTACAACCTTGAAGTGTAU 52 000321_19_PCRleft TCTGGGGGAAAGAAAAGAGTGGU 53 000321_20_PCRleft AAAGAAATAACTCTGTAGATTAAACCTTTCTTTU 54 000321_21_PCRleft TTTCCTTTATAATATGTGCTTCTTACCAGU 55 000321_22_PCRleft TCTTCATGCAGAGACTGAAAACAAAU 56 000321_23_PCRleft TTTGGTATTCCTAATAGTTCAGAATGATGU 57 000321_24_PCRleft CTTTGCCTGATTTTTGACACACCU 58 000321_25_PCRleft AATAGCATAAAGTAAGTCATCGAAAGCAU 59 000321_26_PCRleft TGTCAAATACTAGAATGAAGACCACTGCU 60 000546_00_PCRleft GTCTCAGACACTGGCATGGTGU 61 000546_01_PCRleft CATTTTCAGACCTATGGAAACTGTGAGU 62 000546_02_PCRleft ACAACGTTCTGGTAAGGACAAGGGU 63 000546_03_PCRleft AGGTGCTTACGCATGTTTGTTTCTU 64 000546_04_PCRleft AGTCACAGCACATGACGGAGGTU 65 000546_05_PCRleft TGAGCTGAGATCACGCCACU 66 000546_06_PCRleft CTCCAGAAAGGACAAGGGTGGU 67 000546_07_PCRleft TATCACCTTTCCTTGCCTCTTTCCU 68 000546_08_PCRleft TACTTACTTCTCCCCCTCCTCTGTU 69 000546_09_PCRleft CACCATCTTGATTTGAATTCCCGU 70 000551_00_PCRleft CGAGCGCGTTCCATCCTCU 71 000551_01_PCRleft CCCAAAGTGCTGGGATTACAGGU 72 000551_02_PCRleft AAGCCTCTTGTTCGTTCCTTGTACU 73 007304_00_PCRleft GGTTTGTATTATTCTAAAACCTTCCAAATCTU 74 007304_01_PCRleft TTATTGAGCCTCATTTATTTTCTTTTTCU 75 007304_02_PCRleft GCTCTTAAGGGCAGTTGTGAGATTAU 76 007304_03_PCRleft TGCTGAGTGTGTTTCTCAAACAATTU 77 007304_04_PCRleft TCACAGGTAACCTTAATGCATTGTCTU 78 007304_05_PCRleft TCTTCAGGAGGAAAAGCACAGAACU 79 007304_06_PCRleft TTAACTAGCATTGTACCTGCCACAGU 80 007304_07_PCRleft AAAGGAGAGAGCAGCTTTCACTAACU 81 007304_08_PCRleft TGACAATTCAGTTTTTGAGTACCTTGTU 82 007304_09_PCRleft CCAAAGCAAGGAATTTAATCATTTTGU 83 007304_10_PCRleft ATTTTCTTGGTGCCATTTATCGTTU 84 007304_11_PCRleft TCACTATCAGAACAAAGCAGTAAAGTAGATU 85 007304_12_PCRleft TGATCTCTCTGACATGAGCTGTTTCAU 86 007304_13_PCRleft TGTGTAAATTAAACTTCTCCCATTCCTU 87 007304_14_PCRleft GTAGAACGTGCAGGATTGCTACAU 88 007304_15_PCRleft AAATCCAGATTGATCTTGGGAGTGU 89 007304_16_PCRleft AGCCTTATTAAAGGGCTGTGGCTTU 90 007304_17_PCRleft CTAGGATTACAGGGGTGAGCCACU 91 007304_18_PCRleft ATTTTCCTTCTCTCCATTCCCCTGU 92 007304_19_PCRleft CCTTCATCCGGAGAGTGTAGGGU 93 007304_20_PCRleft TCCTACTTTGACACTTTGAATGCTCTU 94 007304_21_PCRleft TTGACACTAATCTCTGCTTGTGTTCTCU 95 000038_00_PCRright AAUGGAUAAACUACAATUAAAAGUCACAGUCU 96 000038_01_PCRright CACCCAAAUCGAGAGAAGCUGUACU 97 000038_02_PCRright CACAAGGCAAUGUTUACUAUAUGAAGAAAAGU 98 000038_03_PCRright AAAGUTUCAAAUAAGTUGUACUGCCAAGU 99 000038_04_PCRright TUCGCUGUTTUAUCACTUAGAAACAAGU 100 000038_05_PCRright UACCCACAAACAAGAAAGGCAAUTU 101 000038_06_PCRright GACAGCACATUGGUACUGAAUGCTU 102 000038_07_PCRright CCCAAAAUGCUGGGATUACAGGU 103 000038_08_PCRright UTUCUGUTUAAAAAUTUCACAUTUGCTU 104 000038_09_PCRright CAGAGGAAGCAGCUGAUAACAGAAGU 105 000038_10_PCRright GCGAAUGUGAAGCACAGGUTTUUAU 106 000038_11_PCRright GGCUGAAGUGGGAGGATUGCU 107 000038_12_PCRright UGAAUAAUACACAGGUAAGAAATUAGGAAAUCU 108 000038_13_PCRright GCTUAAAACUTUCAUGATUAUAUAAAACATUGCU 109 000249_00_PCRright GCAUGCGCUGUACAUGCCUCU 110 000249_01_PCRright GCCUAGUTUCCAGAACAGAGAAAGGU 111 000249_02_PCRright GGAGGAUAUTTUACACAUTUCTUGAAUCUTU 112 000249_03_PCRright CACUGGUGTUGAGACAGGATUACUCU 113 000249_04_PCRright GCTUCAACAAUTUACUCUCCCAUGU 114 000249_05_PCRright UCUCAGAGACCCACUCCCAGAU 115 000249_06_PCRright GGCUGAGACUGAAACAUCAUAACCTU 116 000249_07_PCRright CAAAUCUGAAGCAUAAAACAAGCCU 117 000249_08_PCRright UTUCCAUGGUCCCAUAAAATUCCCU 118 000249_09_PCRright CUGUAAGAAGGGACAGAACAUCCTU 119 000249_10_PCRright AAUAACAGGCAAAAAUCUGGGCUCU 120 000249_11_PCRright GCUGUACUTTUCCCAAAAGGCCAU 121 000249_12_PCRright AAACCTUGGCAGTUGAGGCCCUAU 122 000249_13_PCRright GGAUTUGAAACCACAUGUGUCUGACU 123 000249_14_PCRright GAAAUTUCAGAAGUGAAAAGGAUCUAAACU 124 000249_15_PCRright ACCCCAAGTUAUCUGCCCACCU 125 000249_16_PCRright AAAGGGUGGUCAUTUGCCCUTU 126 000249_17_PCRright TUGUAUGAGGUCCUGUCCUAGUCCU 127 000249_18_PCRright UCGGAAUACAGAGAAAGAAGAACACAU 128 000321_00_PCRright ACGGCGGCUCUGCUCGCU 129 000321_01_PCRright TUCAAUTTUUGUAUAGUGAUTUGAAGTUGTU 130 000321_02_PCRright TUGAGAGGAAAAUCCAGAATUCGTU 131 000321_03_PCRright UGAGCUAACATUAAAAGGGACAAGUCU 132 000321_04_PCRright UCUACACAGGACTUAAAUCUAUGGGCTU 133 000321_05_PCRright GCAGAGAAUGAGGGAGGAGUACATU 134 000321_06_PCRright AUCAUCCUGUCAGCCTUAGAACCAU 135 000321_07_PCRright AAAAACAUGCUCAUAACAAAAGAAGUAAAU 136 000321_08_PCRright GACAATUAUCCUCCCUCCACAGUCU 137 000321_09_PCRright CCUAUAUCUAAAGCAAAUCAAUCAAAUAUACCAU 138 000321_10_PCRright UGAAUACAUAAAGAAACGUGAACAAAUCU 139 000321_11_PCRright UCAAGUTUCUTUGCCAAGAUATUACAAUAAAUAAU 140 000321_12_PCRright CGAACUGGAAAGAUGCUGCUTTUAAU 141 000321_14_PCRright AGCGCACGCCAAUAAAGACAU 142 000321_15_PCRright GCATUCCTUCUCCTUAACCUCACACU 143 000321_16_PCRright AGAUGTUAAGAAACACCUCUCACUAACAAU 144 000321_17_PCRright UGCAGUTUGAAUGGUCAACAUAACAU 145 000321_18_PCRright AACAUGAUTUGAACCCAGUCAGCCU 146 000321_19_PCRright GAGGAGAGAAGGUGAAGUGCTUGAU 147 000321_20_PCRright UGAATUACCUAUGTUAUGTUAUGGAUAUGGAUTUAU 148 000321_21_PCRright AAGGGCTUCGAGGAAUGUGAGGUAU 149 000321_22_PCRright UCAAAAUAAUCCCCCUCUCATUCUTU 150 000321_23_PCRright UAUGCAAUAUGCCUGGAUGAGGUGU 151 000321_24_PCRright AACTUGGCAUGAAAGAAATUGGUAU 152 000321_25_PCRright AAACAAACCUGCCAACUGAAGAAAU 153 000321_26_PCRright UGUGAGAGACAAUGAAUCCAGAGGU 154 000546_00_PCRright ACAGGUCUCUGCUAGGGGGCU 155 000546_01_PCRright GACAGCAUCAAAUCAUCCATUGCU 156 000546_02_PCRright UCCCAAAGTUCCAAACAAAAGAAAU 157 000546_03_PCRright GCAAAUTUCCTUCCACUCGGAU 158 000546_04_PCRright CUCCUCCCAGAGACCCCAGTU 159 000546_05_PCRright GGUCAGAGGCAAGCAGAGGCU 160 000546_06_PCRright GAAUCUGAGGCAUAACUGCACCCU 161 000546_07_PCRright AGCUACAACCAGGAGCCATUGUCTU 162 000546_08_PCRright CAACCUAGGAAGGCAGGGGAGU 163 000546_09_PCRright CGGGACAAAGCAAAUGGAAGU 164 000551_00_PCRright CTUCAGACCGUGCUAUCGUCCCU 165 000551_01_PCRright AAAGATUGGAUAACGUGCCUGACAU 166 000551_02_PCRright GAAACUAAGGAAGGAACCAGUCCUGU 167 007304_00_PCRright CCCAAATUAAUACACUCTUGUGCUGACU 168 007304_01_PCRright UGGAGCCACAUAACACATUCAAACU 169 007304_02_PCRright TUCUACUTTUUCCUACUGUGGTUGCTU 170 007304_03_PCRright AGCACTUGAGUGUCATUCTUGGGAU 171 007304_04_PCRright GGCUAAGGCAGGAGGACUGCTU 172 007304_05_PCRright UCACCAUAGGGCUCAUAAAATUCACU 173 007304_06_PCRright GGAAAAUACCAGCTUCAUAGACAAAGGU 174 007304_07_PCRright AACUCUGCCAAGAGAUTTUGUGGGU 175 007304_08_PCRright GCUGUAAUGAGCUGGCAUGAGUAUTU 176 007304_09_PCRright TUGUGCCATUAATUCAAAGAGAUGAU 177 007304_10_PCRright AAGGCUCCAUAATUACCCAUGUGCU 178 007304_11_PCRright CCACAGCAUCUTUACATUGAUGUTUCU 179 007304_12_PCRright UGUTUGTUCCAAUACAGCAGAUGAAAU 180 007304_13_PCRright UGTUGTUAAGUCTUAGUCATUAGGGAGAUACAU 181 007304_14_PCRright CAAAGUGCUGCGATUACAGGCAU 182 007304_15_PCRright GGUGUAAAAAUGCAATUCUGAGGUGTU 183 007304_16_PCRright UTUGUGCATUGTUAAGGAAAGUGGU 184 007304_17_PCRright GGUGGGGUGAGAUTTUUGUCAACTU 185 007304_18_PCRright UCCACUAUGUAAGACAAAGGCUGGU 186 007304_19_PCRright GAGGCUACAGUAGGGGCAUCCAU 187 007304_20_PCRright CAAAAGGACCCCAUAUAGCACAGGU 188 007304_21_PCRright GGGGUCCUGUGGCUCUGUACCU

TABLE B Nested Patch SEQ ID. NO. Oligo Name Sequence 189 000038_00_PP L TTAGTGGCTGCTTGTTTTTAAAGAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 190 000038_00_PP R CAAGCAGAAGACGGCATACGATGATACCTTCATATTAGATGCCTCAGT 191 000038_01_PP L TTTCTTGACATTTAAGTATGCTGAGAAAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 192 000038_01_PP R CAAGCAGAAGACGGCATACGATGGATCTACACACCTAAAGATGACA 193 000038_02_PP L GCTTTAAGCAGTCTAAAATATTCTTAATGTTATATTATTTTAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 194 000038_02_PP R CAAGCAGAAGACGGCATACGATACCTCTCTTTCTCAAGTTCTTCTAAATATC 195 000038_03_PP L AAGACTGCAGAAGAGCAATACTTACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 196 000038_03_PP R CAAGCAGAAGACGGCATACGATACTTACATTTTCAGTTAAAGGAAGACTATCT 197 000038_04_PP L CCAATAAAGAAAATGAATAAGCAAATACGTCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 198 000038_04_PP R CAAGCAGAAGACGGCATACGAAACTTACCTGTGCTCGTTTTTCCAT 199 000038_05_PP L TACTATGGCTACCACTTAAAAGCTCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 200 000038_05_PP R CAAGCAGAAGACGGCATACGAACTAACCTCTGCTTCTGTTGCTTG 201 000038_06_PP L ACATCAGTACATGCAAAAATGGTGTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 202 000038_06_PP R CAAGCAGAAGACGGCATACGACTGGAAATATGCATTCAGGACTAAGA 203 000038_07_PP L ACTCCAAATGAAGTGTCTGTATGATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 204 000038_07_PP R CAAGCAGAAGACGGCATACGAGTGAGCCACTGCACCTGG 205 000038_08_PP L CACCTGTGGGCCAAATGAGTTTAGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 206 000038_08_PP R CAAGCAGAAGACGGCATACGATGAAACATGCACTACGATGTACACT 207 000038_09_PP L GCAGGGATCACTAATATAACCCTAATTCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 208 000038_09_PP R CAAGCAGAAGACGGCATACGATGGTGGCCTTATATCCTAATTCATC 209 000038_10_PP L TGGCCTGTAGTCCCCCTAATTTAAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 210 000038_10_PP R CAAGCAGAAGACGGCATACGACAGTCATTGTTTAATGAGGAGAGTGA 211 000038_11_PP L GCCTGTAAATTAAATACAGAATAGAGGATCATTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 212 000038_11_PP R CAAGCAGAAGACGGCATACGATGAACCCTGGAGGCAGAGG 213 000038_12_PP L GAAATTCTGGCTAGCCGTGGTGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 214 000038_12_PP R CAAGCAGAAGACGGCATACGACATGGCTAAAAGAAGGCAGCAAAAA 215 000038_13_PP L AGTAAGAAACAGAATATGGGTCATCTAATTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 216 000038_13_PP R CAAGCAGAAGACGGCATACGATACAATTAGGTCTTTTTGAGAGTATGAATTC 217 000249_00_PP L TGGCGCCAGAAGAGCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 218 000249_00_PP R CAAGCAGAAGACGGCATACGAGCCCGGGCAAAGAGGC 219 000249_01_PP L CTCCAAATACAAACAATAGTGCCTCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 220 000249_01_PP R CAAGCAGAAGACGGCATACGACCTGACTCTTCCATGAAGCGC 221 000249_02_PP L ATGTTACTCATTTTTCCAAATCTCTTTGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 222 000249_02_PP R CAAGCAGAAGACGGCATACGAAGCTTACCTCACCTCGAAAGCC 223 000249_03_PP L TCACCCACTGTCACCTCACCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 224 000249_03_PP R CAAGCAGAAGACGGCATACGAGAGACCTAGGCAAAAAATACATTTCAG 225 000249_04_PP L ATCCAGTAGAGAGATAGATACTAATCCCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 226 000249_04_PP R CAAGCAGAAGACGGCATACGAACCATTCTTACCGTGATCTGGGTC 227 000249_05_PP L AAATAAAACCCAAGATGTCCTGGCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 228 000249_05_PP R CAAGCAGAAGACGGCATACGATTTGGACTGTACCTGCCAACAACT 229 000249_06_PP L CAAAAGAGTAAGAAAAGAGTTGCCAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 230 000249_06_PP R CAAGCAGAAGACGGCATACGAATCTCCACCAGCAAACTATTAAAAATC 231 000249_07_PP L CAGCTACTGTCTCTCCTTGCTGATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 232 000249_07_PP R CAAGCAGAAGACGGCATACGAGTGTATTTGACTAAAGCAAACTCTTAACA 233 000249_08_PP L TTTGTGAAATGAGGGCCCCGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 234 000249_08_PP R CAAGCAGAAGACGGCATACGAGTGGGTGTTTCCTGTGAGTGGAT 235 000249_09_PP L GGGGTGAGGTCACAGGTGTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 236 000249_09_PP R CAAGCAGAAGACGGCATACGATTGCCAGTGGTGTATGGGATTCA 237 000249_10_PP L AGGGGGAGAAAAAGCCCACATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 238 000249_10_PP R CAAGCAGAAGACGGCATACGACACGTCTGGCCGGGC 239 000249_11_PP L AGTGGAGAGACTCAGAATAAGAAGTATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 240 000249_11_PP R CAAGCAGAAGACGGCATACGAACCTGGGGTTGCTGGAAGTAGG 241 000249_12_PP L GTTGCATTTTGGAGGAGCAAGCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 242 000249_12_PP R CAAGCAGAAGACGGCATACGAGCATCCCAGGCAGGCC 243 000249_13_PP L AAGCACCAGGCACCAGAACTAGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 244 000249_13_PP R CAAGCAGAAGACGGCATACGACCAAAGCCTGTGCCCTCC 245 000249_14_PP L AACCAGTTGGGACAAAATGGGAGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 246 000249_14_PP R CAAGCAGAAGACGGCATACGATACCGATAACCTGAGAACACCAAAA 247 000249_15_PP L CGGTGCTGGCTCCTAGGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 248 000249_15_PP R CAAGCAGAAGACGGCATACGACAGCCTCCCAAAGTGCTGG 249 000249_16_PP L GCCTTGTGCTCCTATCTGCCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 250 000249_16_PP R CAAGCAGAAGACGGCATACGACCCTCCAGCACACATGCATG 251 000249_17_PP L TGTGATACTTTAGGCGTTAAAACTGTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 252 000249_17_PP R CAAGCAGAAGACGGCATACGAGGGGTGCCAGTGTGCATC 253 000249_18_PP L GCCTCCCTGTTTGCATCCCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 254 000249_18_PP R CAAGCAGAAGACGGCATACGACCCACAGTGCATAAATAACCATATTT 255 000321_00_PP L AACTGAGCGCCGCGTCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 256 000321_00_PP R CAAGCAGAAGACGGCATACGACACCTGACGAGAGGCAGGTC 257 000321_01_PP L TGTTTCAATAGTTTGCACATAACACTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 258 000321_01_PP R CAAGCAGAAGACGGCATACGATTTAAAATGAGAAAAAAAAATTTCAAAACGTTTTAAG 259 000321_02_PP L TTTCTTATTCAGCATACAAAATAAATGTTTGTAATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 260 000321_02_PP R CAAGCAGAAGACGGCATACGATCCTTTTATGGCAGAGGCTTATATT 261 000321_03_PP L TTCAATTCAAAAGATTATCAGCTCTACATCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 262 000321_03_PP R CAAGCAGAAGACGGCATACGAAAGAATTAATACTTACTAACTTTACTAAATGTGTTAAATAATT 263 000321_04_PP L TTTTTAACATTTTTTCGTAATTTAGAAGTCATAGTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 264 000321_04_PP R CAAGCAGAAGACGGCATACGAAATTTATGAAGTAGCCTGCTATAATCGA 265 000321_05_PP L TGTATCACTGAAAGAAAGTTTTCCAGATATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 266 000321_05_PP R CAAGCAGAAGACGGCATACGAACTCAATAAAAATTGGGGAATTTAGTCC 267 000321_06_PP L CGCAGGGTAGAGTATATCCATAAATTTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 268 000321_06_PP R CAAGCAGAAGACGGCATACGAGTTTGGTACCCACTAGACATTCAAT 269 000321_07_PP L ATGGGTATAACAGCTGTTTCTGTAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 270 000321_07_PP R CAAGCAGAAGACGGCATACGAATTGTTAGGGAGAACTTACATCTAAATCT 271 000321_08_PP L CTTGACTCTTGAACAATGCAGGGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 272 000321_08_PP R CAAGCAGAAGACGGCATACGACAAAACATTAATATTTTATTAAATTTCCTTTCAGATTACC 273 000321_09_PP L CATGTCATTACATCTCTCAGCACACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 274 000321_09_PP R CAAGCAGAAGACGGCATACGAGTGCAATACCTGTCTATAGAATCAGT 275 000321_10_PP L TGCTTTATGCATCAAAAAAGCAGTATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 276 000321_10_PP R CAAGCAGAAGACGGCATACGAGAAACACTATAAAGCCATGAATAACAAAATT 277 000321_11_PP L CACTGCCTCCCACTTGTCTCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 278 000321_11_PP R CAAGCAGAAGACGGCATACGAGTTTCATATATGGCTTACGTTAAAATAGGA 279 000321_12_PP L TTTTGGATTCACTGTGCAGTTCTTTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 280 000321_12_PP R CAAGCAGAAGACGGCATACGAATTATTACTCTATAGTACCACGAATTACAATGA 281 000321_14_PP L TTGCCAGGCTGGGGTGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 282 000321_14_PP R CAAGCAGAAGACGGCATACGAATGAAAAATGTTGTCATTCAGAAGTTTGC 283 000321_15_PP L ACTAAAAGTAAAAAATTTACCTAAAATTTTGAATGGATAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 284 000321_15_PP R CAAGCAGAAGACGGCATACGAATCCCTCTCCCCCGACCA 285 000321_16_PP L TGAGCTAGGTATTTTTTTGGAAGTTATTATCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 286 000321_16_PP R CAAGCAGAAGACGGCATACGAAATTTGTTAGCCATATGCACATGAA 287 000321_17_PP L AGTACTATGAATTTTAGGCACAATTGACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 288 000321_17_PP R CAAGCAGAAGACGGCATACGAATATTTTGCTTACATATCTGCTGCAG 289 000321_18_PP L CAAGTTGGCTAAGAATCACAGATTATACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 290 000321_18_PP R CAAGCAGAAGACGGCATACGAAGTTTCAGAGTCCATGCTCTTGAAA 291 000321_19_PP L GTAGCATTTTAACAGAAACCTCTTTTCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 292 000321_19_PP R CAAGCAGAAGACGGCATACGATTTCTTACTTGGTCCAAATGCCTGT 293 000321_20_PP L AATACCATTTTCTTTCTTTTAGCCTCAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 294 000321_20_PP R CAAGCAGAAGACGGCATACGACAAAAAAACTTACTATGGAAAATTACCTACCT 295 000321_21_PP L ACCTTTAGATTTTCTTTTCTAATAGTTTATAATACTTTTTGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 296 000321_21_PP R CAAGCAGAAGACGGCATACGATGGTGACAAGGTAGGGGGC 297 000321_22_PP L CCTGGTGGAAGCATACTGCAAAATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 298 000321_22_PP R CAAGCAGAAGACGGCATACGAACTACTTCCCTAAAGAGAAAACACAC 299 000321_23_PP L ACAATTTTGCAGAGATGAGCATAAATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 300 000321_23_PP R CAAGCAGAAGACGGCATACGATTGAATAACTGCATTTGGAAATTCAAATTAT 301 000321_24_PP L CATAGTTAGCAACCTCAAGTTATAGTTTGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 302 000321_24_PP R CAAGCAGAAGACGGCATACGAAAGCCAGGAGCAGTGCTGA 303 000321_25_PP L TGGAAAACTCAAATTTCCAGTAACTATGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 304 000321_25_PP R CAAGCAGAAGACGGCATACGATATACATTCTTTTATATAACGAAAAGACTTCTTGC 305 000321_26_PP L GCGCTCAGGACCTTGCAAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 306 000321_26_PP R CAAGCAGAAGACGGCATACGAGTACACAGTGTCCACCAAGGTC 307 000546_00_PP L GGAACCCCCTCCCCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 308 000546_00_PP R CAAGCAGAAGACGGCATACGAGGGGTTGGGGTGGGG 309 000546_01_PP L CCTGCCCTTCCAATGGATCCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 310 000546_01_PP R CAAGCAGAAGACGGCATACGATGGGACGGCAAGGGGG 311 000546_02_PP L CCAGGTCCCCAGCCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 312 000546_02_PP R CAAGCAGAAGACGGCATACGAGCAGGGGGATACGGCCA 313 000546_03_PP L CAACTGGAAGACGGCAGCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 314 000546_03_PP R CAAGCAGAAGACGGCATACGAAAGATGCTGAGGAGGGGCC 315 000546_04_PP L GGGGCAGCGCCTCACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 316 000546_04_PP R CAAGCAGAAGACGGCATACGAGCAAACCAGACCTCAGGCG 317 000546_05_PP L GCCCAGGCTGGAGTGCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 318 000546_05_PP R CAAGCAGAAGACGGCATACGAGGGGCACAGCAGGCC 319 000546_06_PP L ACCAGGCTCCATCTACTCCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 320 000546_06_PP R CAAGCAGAAGACGGCATACGATGGTCTCCTCCACCGCTTC 321 000546_07_PP L GGTGTTGTTGGGCAGTGCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 322 000546_07_PP R CAAGCAGAAGACGGCATACGATGAGGCATCACTGCCCCC 323 000546_08_PP L GCCCACGGATCTGCAGCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 324 000546_08_PP R CAAGCAGAAGACGGCATACGAAGGGCCAGGAAGGGGC 325 000546_09_PP L GGGCCTAAGGCTGGGACAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 326 000546_09_PP R CAAGCAGAAGACGGCATACGACCTGGGTGCTTCTGACGC 327 000551_00_PP L TCTTCGCGCGCGCTCGGTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 328 000551_00_PP R CAAGCAGAAGACGGCATACGAGCTGGGTCGGGCCTAAG 329 000551_01_PP L GGCACGGTGGCCCACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 330 000551_01_PP R CAAGCAGAAGACGGCATACGACAGGCAAAAATTGAGAACTGGGCTT 331 000551_02_PP L CTCAGTGGCAGACTAGGGTCTCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 332 000551_02_PP R CAAGCAGAAGACGGCATACGAATCTAGATCAAGACTCATCAGTACCA 333 007304_00_PP L ATGACAACTTCATTTTATCATTTTAAAATAAAGTAAATTTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 334 007304_00_PP R CAAGCAGAAGACGGCATACGATACCAGATGGGACACTCTAAGATTT 335 007304_01_PP L CTAGCAGGGTAGGGGGGGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 336 007304_01_PP R CAAGCAGAAGACGGCATACGATACTTGCAAAATATGTGGTCACACT 337 007304_02_PP L TCAAAAGGCAAATAGCCATGAAAAGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 338 007304_02_PP R CAAGCAGAAGACGGCATACGACCAACCTAGCATCATTACCAAATTATATAC 339 007304_03_PP L TACTTTCTTGTAGGCTCCTGAAATTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 340 007304_03_PP R CAAGCAGAAGACGGCATACGAATTCAACACTTACACTCCAAACCTG 341 007304_04_PP L CCCTATGTATGCTCTTTGTTGTGTTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 342 007304_04_PP R CAAGCAGAAGACGGCATACGACTAGCCTGGGCCACAGAG 343 007304_05_PP L AAGAACAGTCAAGCAATTGTTGGCCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 344 007304_05_PP R CAAGCAGAAGACGGCATACGATCCCAAAGCTGCCTACCACAAATA 345 007304_06_PP L AGATATTCAACTAGAAATATTTACTGAGCATCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 346 007304_06_PP R CAAGCAGAAGACGGCATACGATCTCTTTGACTCACCTGCAATAAGT 347 007304_07_PP L GATTACAGAAAGCTGACCAATCTTATTTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 348 007304_07_PP R CAAGCAGAAGACGGCATACGATGTAAAGGTCCCAAATGGTCTTCAG 349 007304_08_PP L TCACAAGCAGCTGAAAATATACAAAAATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 350 007304_08_PP R CAAGCAGAAGACGGCATACGAGTGCCACATGGCTCCACATG 351 007304_09_PP L AGGACTGGATTTACTTTCATGTCACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 352 007304_09_PP R CAAGCAGAAGACGGCATACGAGTCAGCAAACCTAAGAATGTGGGAT 353 007304_10_PP L TTGCATGGTATCCCTCTGCTTCAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 354 007304_10_PP R CAAGCAGAAGACGGCATACGAGAGCAAGGATCATAAAATGTTGGAG 355 007304_11_PP L ACTGCTTTAAATGGAATGAGAAAACAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 356 007304_11_PP R CAAGCAGAAGACGGCATACGATACCTTTCCACTCCTGGTTCTTTAT 357 007304_12_PP L GCTGGGCAGCCAAAGCATAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 358 007304_12_PP R CAAGCAGAAGACGGCATACGAATTACCTAGATCTTGCCTTGGCAAG 359 007304_13_PP L CAGGTAAGGGGTTCCCTCTGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 360 007304_13_PP R CAAGCAGAAGACGGCATACGAATGGATACACTCACAAATTCTTCTGG 361 007304_14_PP L ATTCCACCATGGCATATGTTTACCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 362 007304_14_PP R CAAGCAGAAGACGGCATACGAGCGCCACCGTGCCTC 363 007304_15_PP L AGAAGCTAAAGAGCCTCAGTTTTTTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 364 007304_15_PP R CAAGCAGAAGACGGCATACGAAAAGGGAGGAGGGGAGAAATAGTAT 365 007304_16_PP L CAGAGGAGAGGTCCTTCCCTCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 366 007304_16_PP R CAAGCAGAAGACGGCATACGAGCATTGATGGAAGGAAGCAAATACA 367 007304_17_PP L CATTCAGGCCAGGCGCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 368 007304_17_PP R CAAGCAGAAGACGGCATACGAGAGGGAGGGAGCTTTACCTTTCTG 369 007304_18_PP L TGGAAGAAGAGAGGAAGAGAGAGGGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 370 007304_18_PP R CAAGCAGAAGACGGCATACGAGCTGGAACTCTGGGGTTCTCC 371 007304_19_PP L GCATACTTAACCCAGGCCCTCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 372 007304_19_PP R CAAGCAGAAGACGGCATACGAAGGGACTGACAGGTGCCAG 373 007304_20_PP L CCTGGATCCCCAGGAAGGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 374 007304_20_PP R CAAGCAGAAGACGGCATACGAACATGCAGGCACCTTACCATG 375 007304_21_PP L CATCTGCCCAATTGCTGGAGACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT 376 007304_21_PP R CAAGCAGAAGACGGCATACGAGTGGCTGGCTGCAGTCAG

TABLE C SEQ ID. NO. Oligo Name Sequence 377 Upstream Universal Primer For Ligation ACACTCTTTCCCTACACGACGCTCTTCCGATC 378 Downstream Universal Primer For 5′Phosphate TCGTATGCCGTCTTCTGCTTG 3′ Ligation 379 Final Universal PCR Barcode Forward GCCTCCCTCGCGCCATCAGCTACACGACGCTCTTCCGATC Primer for Normal Sample 380 Final Universal PCR Barcode Reverse GCCTTGCCAGCCCGCTCAGCAAGCAGAAGACGGCATACGA Primer for Normal Sample 381 Final Universal PCR Barcode Forward GCCTCCCTCGCGCCATCAGGTCACACTACACGACGCTCTTCCGATC Primer for Colon Cancer Sample 382 Final Universal PCR Barcode Reverse GCCTTGCCAGCCCGCTCAGCAGTCACAAGCAGAAGACGGCATACGA Primer for Colon Cancer Sample

Nucleic Acid Patch PCR

Genomic DNA from a moderately differentiated colon adenocarcinoma primary tumor and adjacent normal tissue from an 81-year-old male (Biochain catalog #D8235090-PP-10) was used as template for the first PCR. Targets were amplified in a reaction containing 1 μg human genomic DNA, 50 nM each of 94 Forward PCR primers, 50 nM each of 94 Reverse PCR primers, 5 units of AmpliTaq Polymerase Stoffel Fragment (Applied Biosystems), 200 μM each dNTP, 2 mM MgCl₂, 20 mM Tris-HCl pH 8.4 and 50 mM KCl in a total volume of 10 μl. This reaction was incubated at 94° C. for 2 min followed by (94° C. for 30 sec, 56° C. for 30 sec, 72° C. for 6 min)×10 cycles, and then held at 4° C.

To prepare for the next round of oligonucleotide hybridization, the uracil-containing primers from the first reaction were cleaved from the amplicons by the addition of 1 unit heat labile Uracil-DNA Glycosylase (USB), 10 units of Endonuclease VIII (NEB), and 10 units of Exonuclease I (USB). This mix was incubated at 37° C. for 2 hours followed by heat inactivation at 95° C. for 20 minutes, and held at 4° C. To remove the unincorporated nucleotide from the mix, 0.05 U Apyrase (NEB) was added to the reaction and incubated at 30° C. for 30 minutes.

Nucleic acid patch-driven ligation of the universal primers to correct amplicons is performed by addition of more reactants to the initial tube to result in the following final concentrations: 20 nM each nucleic acid patch oligonucleotide, 40 nM Universal Primer 1, 40 nM Universal Primer 2 with 5′ phosphate and 3′ three carbon spacer, 5 U Ampligase (Epicentre), and 1× Ampligase Reaction Buffer (Epicentre) in a total volume of 25 μl. This reaction was incubated at 95° C. for 15 min followed by (94° C. for 30 sec, 65° C. for 2 min, 55° C. for 1 min, 60° C. for 5 min) for 100 cycles, and held at 4° C.

Incorrect products, template genomic DNA and excess primer were degraded by the addition of 10 U Exonuclease I (USB) and 200 U Exonuclease III (Epicentre). This mix was incubated at 37° C. for 2 hours followed by heat inactivation at 95° C. for 20 minutes, and held at 4° C. Each selection reaction was purified using a Qiaquick Spin Column (Qiagen) and the final elution was performed with 30 μl elution buffer (EB).

For the final PCR using the universal primers, reagents were added to the elution to result in these final concentrations in 50 μl: 0.5 μM each Tailed Universal Primer (see below), 10 U Platinum Taq Polymerase (Invitrogen) 0.5 mM each dNTP, 2 mM MgCl₂, 0.5 M Betaine to improve the amplification of GC-rich sequences, 20 mM Tris-HCl pH 8.4 and 50 mM KCl. This reaction was incubated at 93° C. for 2 min followed by (93° C. for 30 sec, 60° C. for 6 min) for 27 cycles, and held at 4° C. The universal PCR used the Final Universal PCR primers tailed with 454 Life Sciences A or B oligonucleotide at the 5′ end, followed by a sample-specific 6 bp sequence and ending at the 3′ end with the same universal primer sequence ligated to the amplicons in the nucleic acid patch PCR procedure. The PCR product smear between the expected sizes was confirmed by running on a 3% Metaphor Agarose gel (Lonza). The reactions were then purified on a Qiaquick Spin Column (Qiagen). The eluted DNA was quantified on a Nanodrop spectrophotometer (ThermoFisher Scientific Inc.), and the same quantity of DNA was pooled together from the two separate samples. This pooled sample was sequenced using the 454 sequencing system on the 454 Life Sciences/Roche FLX machine.

Sequence Analysis

To determine which sequences matched the intended targets, the reads were aligned against a database of reference target sequences for each target using the BLASTN software at the Washington university in St. Louis BLAST archives (http://blast.wustl.edu). The number of reads that matched significantly to each exon was determined (p<0.02). The first six bases of sequence from each read, the sample specific DNA barcode, was used to determine whether the sequence came from the tumor sample or the normal sample. The number of reads that did not match targeted sequence was determined, and those sequences were aligned to a database of nucleic acid patch oligonucleotide sequence to identify what fraction was due to primer artifacts. For each exon, CLUSTALW was used to generate a multiple sequence alignment of all of the reads against the reference sequence (Larkin, Blackshields, Brown, Chema, McGettigan, McWilliam, Valentin, Wallace, Wilm, Lopez et al. 2007). The majority of the differences from the reference sequence were insertion or deletion mutations (indels) adjacent to stretches of identical nucleotides (homopolymers), which is a known error-prone feature for 454 sequencing (Ronaghi, Uhlen and Nyren 1998). To filter these out, all the positions that did not match the reference sequence but were in greater than 30% of the reads were examined.

Results

Oligonucleotides were designed for 94 of the 96 exons from the six nucleotide sequences encoding colon-cancer related proteins. Attempts to design oligonucleotides to two of the 96 exons failed; the last exon of APC failed because of length (˜6000 bp) and an exon in RB1 failed due to the presence of Alu repeat elements surrounding the exon.

55,068 sequencing reads were obtained. At least one read from each sample was mapped to 90 of the 94 exons (95.7%). The 4 exons that failed to amplify were due to imperfect primer/patch design. Two of the loci could not be amplified in separate individual PCR reactions, indicating PCR primer failure. The other 2 loci failed because their patch oligonucleotides bound to multiple locations in the genome. This problem could be avoided by more careful primer design. Ninety percent of all reads (49553 reads) mapped to one of the targeted exons. Thus, a 125,000-fold enrichment was achieved with nucleic acid patch PCR from genomic DNA (90% specificity×total possible fold enrichment). When selecting a fraction of the genome this small, the total possible enrichment is 138,888 fold (3×10⁹ by genome/21.6 kbp targeted). Of the remaining 10% of reads that did not match the targeted regions, most (85%) appear to be due to concatamers of nucleic acid patch oligonucleotides that contain Alu elements. It is likely that designing oligonucleotides that do not overlap repetitive genomic elements could reduce this background.

These results demonstrate that nucleic acid patch PCR can be performed on multiple samples in parallel, which can then be labeled with sample-specific DNA barcodes and sequenced as a pool. The choice of targets and target boundaries is flexible, and a wide range of sizes can be amplified simultaneously (here, 74 bp to 438 bp). Nucleic acid patch PCR is robust and sensitive, as this method was able to amplify 90 of the 94 targeted exons.

Example 3 Uniformity of Nucleic Acid Patch PCR Per Exon in Each Sample

Ideally for any multiplexed PCR method, all targeted regions would be uniformly amplified within each reaction by all primer pairs, and across samples from different templates. To analyze the uniformity of amplification of the 90 regions generated by nucleic acid patch PCR in Example 2, the number of reads obtained for each targeted was graphed (FIG. 3A). The number of sequencing reads obtained for each exon is also presented numerically in TABLE D. Sequence coverage ranged over 2-3 logs (base 10), with 75% (68/90) of exons having between 10 and 500 reads in both samples (50 fold abundance range). The median number of reads per exon was 145. Seventy-six percent of all exons fell within 5-fold coverage of this median (29-725 reads). There were no parameters found that explain the non-uniformity. Exon non-uniformity did not correlate with the gene, the size of the amplicon, nor the GC content of the oligonucleotides.

TABLE D Reads Per Exon in Tumor and Normal Samples Number of Reads Number of Reads RefSeq_Exon Number in Normal Sample in Tumor Sample NM_000551_1 1 2 NM_000321_22 3 8 NM_000546_5 4 12 NM_007304_0 7 3 NM_007304_2 7 8 NM_000546_3 10 5 NM_000546_9 10 15 NM_000321_16 13 9 NM_007304_4 15 24 NM_000038_8 16 31 NM_000321_20 16 26 NM_000249_8 17 17 NM_000321_9 28 30 NM_000321_26 32 14 NM_000321_17 33 38 NM_007304_10 33 33 NM_000321_14 38 98 NM_000321_24 42 65 NM_000321_1 44 129 NM_000038_2 47 55 NM_000551_2 47 113 NM_000321_7 48 17 NM_000038_7 56 59 NM_000321_2 60 83 NM_000249_17 63 43 NM_000546_8 63 59 NM_007304_21 66 115 NM_000038_11 68 34 NM_000321_4 72 65 NM_000321_5 75 89 NM_000321_6 79 100 NM_000321_10 80 65 NM_000038_12 83 75 NM_000321_18 87 98 NM_007304_7 88 58 NM_007304_9 95 98 NM_007304_14 96 87 NM_007304_16 105 118 NM_000546_0 108 85 NM_000249_1 111 150 NM_000038_13 121 70 NM_007304_8 124 114 NM_000321_11 129 97 NM_000038_5 133 71 NM_000249_12 143 91 NM_000546_1 148 92 NM_007304_15 149 109 NM_000249_7 154 149 NM_007304_20 159 178 NM_007304_19 160 134 NM_007304_13 162 160 NM_007304_11 165 74 NM_000249_10 200 136 NM_000321_3 222 243 NM_000249_0 235 146 NM_007304_17 247 227 NM_000321_15 253 265 NM_000249_15 267 145 NM_000546_6 283 151 NM_007304_12 286 270 NM_000249_2 288 249 NM_000249_9 292 225 NM_000038_4 314 227 NM_000321_19 317 464 NM_000038_3 332 284 NM_000249_13 353 199 NM_000038_9 356 316 NM_000321_25 386 301 NM_000546_7 396 200 NM_000546_4 415 206 NM_000321_21 416 373 NM_007304_5 464 358 NM_000249_18 498 340 NM_000038_10 524 338 NM_007304_18 532 373 NM_000038_0 549 273 NM_000249_6 587 470 NM_000249_3 648 433 NM_000249_4 660 574 NM_000321_12 670 434 NM_000038_1 713 320 NM_007304_3 940 833 NM_000249_14 942 483 NM_000249_16 948 471 NM_007304_6 975 779 NM_000038_6 1170 780 NM_000321_23 1198 967 NM_000321_8 1283 697 NM_007304_1 1932 1605 NM_000249_5 2813 1665

To test the reproducibility of the nucleic acid patch PCR method, the number of reads per exon from the tumor and normal samples were correlated. The correlation was high (R² of 93%), indicating high reproducibility (FIG. 4A). In fact, 85% (77/90) of exons displayed at most a 2 fold difference in abundance between samples, and all exons were within 3 fold relative abundance between samples (FIG. 4B).

These results demonstrate that even though the abundance of PCR products varies between exons, the abundance of each exon is highly reproducible across different reactions and samples.

Example 4 SNP and Mutation Discovery and Validation

The variants from the reference sequence identified by nucleic acid patch PCR and 454 FLX sequencing in Example 2 were validated by performing individual PCR reactions for each variant locus, cloning the amplicons into E. coli, and sequencing 12 clones for each variant. Sequence variants were then analyzed for novelty and whether they affected the translation product of that nucleotide sequence.

Methods

The PCR for each locus in each sample was performed in a total volume of 50 μl. The reaction contained 1×PCR buffer lacking MgCl₂ (Invitrogen, Carlsbad, Calif.), 10 units Platinum Taq Polymerase (Invitrogen Carlsbad, Calif.), 0.5 mM each dNTP, 0.5 M Betaine, 0.5 μM Forward Primer, 0.5 μM Reverse Primer, and 100 ng genomic DNA from either the colon tumor or the adjacent normal tissue (Biochain catalog #D8235090-PP-10). This reaction was incubated at 93° C. for 2 minutes, followed by (93° C. for 30 sec, 55° C. for 6 minutes)×30 cycles, and held at 4° C. One fifth of the PCR reaction was verified by electrophoresis on a 2% agarose gel. The PCR products were ligated into the pGEM-T Easy Vector using Rapid Ligation Buffer according to the manufacturer's instructions (Promega, Madison, Wis.), transformed into GC10 Competent Cells (Gene Choice) and grown overnight on LB-agar (Luria-Broth) plates containing standard concentrations of carbenicillin, X-gal and IPTG. After overnight growth, at least 12 colonies were picked from the plates and added to 50 μl colony PCR reactions containing 1×PCR Reaction Buffer (Sigma, St. Louis, Mo.), 2 units Jumpstart Taq Polymerase (Sigma), 0.2 mM each dNTP, 0.5 μM M13 Forward Primer (5′ CGCCAGGGTTTTCCCAGTCACGAC 3′) (SEQ ID NO:383), 0.5 μM M13 Reverse Primer (5′ TCACACAGGAAA CAGCTATGAC 3′) (SEQ ID NO:384), and 0.01% Tween. The reaction was incubated at 94° C. for 10 minutes, followed by (94° C. for 1 min 30 sec, 55° C. for 1 min, 72° C. for 1 min)×35 cycles, and held at 4° C. These reactions were then treated with 10 μl Exo-SAP to degrade the remaining primers and nucleotides by adding 0.2 units Exonuclease I (USB, Cleveland, Ohio) and 0.2 units Shrimp Alkaline Phosphatase (SAP) (Promega, Madison, Wis.) in 1×SAP buffer (Promega, Madison, Wis.), incubating at 37° C. for 30 min, then by 80° C. for 30 min. The Sanger sequencing/cycle sequencing reactions were 20 ul and contained 1.5 μl Exo-SAP Treated colony PCR, 1 μl Big Dye Terminator v3.1 RR-100 Mix (Applied Biosystems, Foster City, Calif.), 2 mM MgCl₂, and 0.16 μM M13 Forward Primer. They were incubated at 96° C. for 1 min, followed by (96° C. for 10 sec, 50° C. for 5 sec, 60° C. for 4 min)×24 cycles, and held at 4° C. The reactions were ethanol precipitated with sodium acetate and submitted to the Washington University Genome Sequencing Center to load on the ABI 3730 (Applied Biosystems, Foster City, Calif.). Trace files were analyzed using the Phred software (Ewing and Green 1998; Ewing, Hillier, Wendl and Green 1998), and the resulting sequencing reads were aligned to the reference sequence using the BLAT software on the UCSC Genome Browser (Kent 2002; Kent, Sugnet, Furey, Roskin, Pringle, Zahler and Haussler 2002).

Sequence variants for each exon were identified, and the UCSC Genome browser was used to determine the presence of these variants in the NCBI database of SNPs (dbSNP, www.ncbi.nlm.nih.gov/projects/SNP/index.html), and whether they disrupted a codon. To determine if the tumor specific mutation identified in this analysis had been previously reported, the Catalog of Somatic Mutations in Cancer (www.sanger.ac.uk/genetics/CGP/cosmic/) was searched.

Results

Seven variants from the reference sequence were identified (TABLE E). The SNPs and mutations identified by nucleic acid patch PCR and 454 FLX sequencing were validated by performing individual PCR reactions from the original patient samples, cloning the amplicons, and sequencing at least 8 clones per locus using standard Sanger sequencing. Five of these variants were already in the NCBI database of SNPs (dbSNP; http://www.ncbi.nlm.nih.gov/SNP/). The individual sequenced was germline homozygous at three of these SNPs (rs17883323, rs185587, rs3020646) and was germline heterozygous at two other SNPs in the database, rs2229992 and rs351771. The A allele of the SNP rs2229992 was in 54% of reads from the tumor sample and 54% of reads from normal sample. The C allele of the SNP rs351771 was in 48% of reads from the tumor sample and 47% of reads from normal sample. The ability to detect both alleles of these known polymorphisms at near equal frequency indicates that nucleic acid patch PCR provides high allele sensitivity that is reproducible across samples. SNP in an intron of APC that was not yet in dbSNP (rs62626346) was also discovered. The sequenced individual was heterozygous in both the tumor and normal samples at this intronic position. A novel germline SNP was discovered in the sequenced individual in one of the most extensively surveyed genes, APC. This illustrates that medical resequencing of well-characterized candidate genes will yield more insight into genetic variation in individuals.

TABLE E Mutation and SNPs discovered. Bold mutation is tumor specific. Fraction of Reads with Variant Colon Exon Reference Amino Acid Adenocarcinoma Adjacent Normal Protein Ref Seq ID number Location* Base Variant Change Tissue Tissue APC NM_000038 10 rs2229992 T C none 143/301 48% 222/468 47% APC NM_000038 12 rs351771 G A none 37/68 54% 43/79 54% APC NM_000038 12 chr5: 112192485 C T Arg-> 23/68 33%  3/80 4% STOP APC NM_000038 13 rs62626346† T C intronic 17/29 59% 27/50 54% TP53 NM_000546 1 rs17883323 G T intronic 41/41 100% 50/50 100% RB1 NM_000321 11 rs185587 G T intronic 79/79 100% 102/102 100% RB1 NM_000321 24 rs3020646 C T intronic 24/24 100% 18/18 100% *Location is according to the March 2006 human genome assembly from the UCSC Genome Browser †Novel germline SNP

A tumor-specific nonsense mutation was also discovered. It is a C to T substitution in the APC gene at chr5:112192485 that results in a codon for arginine changing to a stop codon. This is likely a significant mutation in this individual's colon tumor because it is a nonsense mutation in a gene that is already known to cause colon cancer. This mutation was in 33% of reads from the tumor sample. This mutation is adjacent to a heterozygous SNP, and we discovered that 62% of the SNP A allele reads had the nonsense mutation, and 0% of the SNP G allele reads had the nonsense mutation. This indicates that the nonsense mutation occurred on the A allele during the clonal expansion of the tumor. This mutation was previously observed in an ovarian endometrioid adenocarcinoma and is Mutation ID #19040 in the Catalog of Somatic Mutations in Cancer (http://www.sanger.ac.uk/genetics/CGP/cosmic/).

In summary, this method has the allele sensitivity necessary for variant discovery in personal genome sequencing since both alleles of heterozygous SNPs were identified at near-even frequencies. Indeed, the utility of nucleic acid patch PCR is best illustrated by the fact that a novel, cancer-specific mutation was discovered in this pilot study.

Example 5 SNP Sensitivity Analysis

To determine the sensitivity of the nucleic acid patch PCR method coupled with 454 sequencing, each exon analyzed in examples 2 to 5 was individually amplified by PCR from the same colon cancer and adjacent normal tissue samples as used above. Direct Sanger sequencing was then performed. The sequences obtained were then compared to sequences generated using nucleic acid patch PCR and 454 sequencing.

The PCR for each locus in each sample was performed in a total volume of 50 ul. The reaction contained 1×PCR Buffer —MgCl2 (Invitrogen, Carlsbad, Calif.), 5 units Platinum Taq Polymerase (Invitrogen Carlsbad, Calif.), 0.5 mM each dNTP, 0.5 M Betaine, 0.5 μM Locus Specific Forward Primer, 0.5 μM Locus Specific Reverse Primer, and 20 ng genomic DNA from the adjacent normal tissue (Biochain catalog #D8235090-PP-10). This reaction was incubated at 93° C. for 2 min, followed by (93° C. for 30 sec, 55° C. for 6 min)×30 cycles, and held at 4° C. One fifth of the PCR reaction was verified by electrophoresis on a 2% agarose gel. These reactions were then treated with 10 μl Exo-SAP to degrade the remaining primers and nucleotides by adding 0.2 units Exonuclease I (USB, Cleveland, Ohio) and 0.2 units Shrimp Alkaline Phosphatase (SAP) (Promega, Madison, Wis.) in 1×SAP buffer (Promega, Madison, Wis.), incubating at 37° C. for 30 min, then by 80° C. for 30 min. The Sanger sequencing/cycle sequencing reactions were 20 μl and contained 1.5 μl ExoSAP-treated individual exon PCR, 1 μl Big Dye Terminator v3.1 RR-100 Mix (Applied Biosystems, Foster City, Calif.), 2 mM MgCl2, and 0.16 μM Forward or Reverse PCR Primer. They were incubated at 96° C. for 1 min, followed by (96° C. for 10 sec, 50° C. for 5 sec, 60° C. for 4 min)×24 cycles, and held at 4° C. The reactions were ethanol precipitated with sodium acetate and submitted to the Washington University Genome Sequencing Center to load on the ABI 3730 (Applied Biosystems, Foster City, Calif.). Trace files from both forward and reverse reads were analyzed for SNPs using PolyPhred and manual inspection (Nickerson, Tobe and Taylor 1997).

No additional SNPs were identified in the DNA sample beyond the six germline SNPs already identified. Thus, in this experiment, the sensitivity of the method is 100%.

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Example 6 Bisulfite Nucleic Acid Patch PCR Proof of Concept

In this example, various features of the method of the invention are demonstrated including: 1. Creating nucleic acid template with defined ends using AluI restriction digest. 2. Treatment with sodium bisulfite to detect DNA methylation by sequencing. 3. Using small quantities of DNA. The method is depicted in FIG. 4.

Template Preparation

Genomic DNA from breast and colon cancer and adjacent normal tissue was digested with the AluI restriction endonuclease in 10 ul total volume reaction containing genomic DNA, 10 U AluI enzyme (NEB), and 1× NEBuffer 2 (NEB). This reaction was incubated at 37° C. for 1 hour, followed by heat inactivation of the enzyme at 65° C. for 20 min, and held at 4° C. until the subsequent step. To demonstrate the efficacy of this method with small quantities of DNA, multiple reactions were performed using decreasing quantities of genomic DNA including 900, 675, 450, 250, 225, 112, 70, 50, 20, 1.6, 0.8, and 0.4 ng genomic DNA. A control reaction lacking genomic DNA was also prepared.

Nucleic Acid Patch Ligation

Nucleic acid patch oligos were designed as described in Example 2 but were designed to anneal adjacent to the AluI restriction enzyme site upstream and downstream of promoters of a select 94 gene in the human genome. These loci were selected because they are the promoters of genes frequently mutated in cancer. Nucleic acid patch driven ligation of the universal primers to selected fragments was performed by addition of more reactants to the initial tube to result in the following final concentrations: 2 nM each nucleic acid patch oligo, 200 nM Universal Primer 1, 200 nM Universal Primer 2 with 5′ phosphate and 3′ three carbon spacer, 5 U Ampligase (Epicentre), and 1× Ampligase Reaction Buffer (Epicentre) in a total volume of 25 ul. This reaction was incubated at 95° C. for 15 minutes followed by (94° C. for 30 sec, 65° C. for 8 min) for 100 cycles, and held at 4° C.

Incorrect products, template genomic DNA and excess primer were degraded as described in Example 2. In brief, 10 U Exonuclease I (USB) and 200 U Exonuclease III (Epicentre) were added to the reaction. This mix was incubated at 37° C. for 1 hour followed by heat inactivation at 95° C. for 20 minutes, and held at 4° C.

Sodium Bisulfite Treatment

The reactions were then treated with sodium bisulfite to convert unmethylated cytosines to uracil. This was achieved by using the EZ DNA Methylation Gold Bisulfite Treatment Kit (Zymo Research) following the manufacture's instructions. Since the sample volume after the exonuclease treatment was 27 ul, the CT Conversion Reagent from the kit was made by adding 830 ul dH2O instead of 900 ul dH2O. The DNA was eluted from the column in the final step with 10 ul M-Elution buffer.

PCR Amplification

The universal primers were then used to PCR amplify the selected bisulfite converted loci from each sample. For the PCR, reagents were added to the last 10 ul column elution to result in these final concentrations in 50 ul: 0.5 uM each tailed Universal Primer, 10 U Platinum Taq Polymerase (Invitrogen), 0.5 mM each dNTP, 2 mM MgCl₂, 0.5M Betaine, 20 mM Tris-HCl pH 8.4 and 50 mM KCl. This reaction was incubated at 93° C. for 2 minutes followed by (93° C. for 30 sec, 57° C. for 6 min) for 29 cycles, and held at 4° C. As described in Example 2, the universal PCR used primers tailed with 454 Life Sciences A or B oligo at the 5′ end, followed by a sample specific DNA sequence and ending at the 3′ end with the nucleic acid patch universal primer sequence. The PCR product smear between the expected sizes was confirmed by running on a 3% Metaphor Agarose gel (Lonza). The reactions were then purified on a Qiaquick Spin Column (Qiagen). An aliquot of the reactions was analyzed by gel electrophoresis on an agarose gel (Lonza).

The eluted DNA of the reactions using 250 ng of genomic DNA was quantified on the Nanodrop (www.nanodrop.com) and the same quantity of DNA was pooled together from each of the separate samples. This pooled sample was submitted for sequencing on the 454 Life Sciences/Roche FLX machine. Sequence and data analysis were as described in Example 2.

Results

Highly multiplexed bisulfite PCR was successful even when small quantities of genomic DNA were used (FIG. 5). The expected smear of products is seen in the lane that contained 900 ng DNA, and the reaction generates the expected products even when as little as 20 ng of genomic DNA is used. Using less than 20 ng of genomic DNA might also have been successful, but the sensitivity of the imaging was not sufficient to reliably detect it.

Sequence analysis of the reactions performed using 250 ng of human tumor genomic DNA demonstrated that 100% of the targeted regions were successfully amplified and sequenced. All of the 94 targeted promoters were sequenced at least once (FIG. 6). The method was also very specific, with 90% of all reads matching the targeted promoters.

In summary, digesting genomic DNA with AluI successfully defined the ends of nucleic acid templates even when a very small quantity of genomic DNA treated with sodium bisulfite was used.

Example 7 Bisulfite Nucleic Acid Patch PCR and Tumor Analysis

Inappropriate CpG DNA methylation has been found in most types of cancers¹. Genes that participate in numerous pathways involved in malignancy can acquire aberrant promoter methylation². Tumor suppressor genes frequently exhibit promoter hypermethylation, an epimutation that is associated with inappropriate gene silencing². A recent study has found that several key tumor suppressor genes exhibit promoter hypermethylation more often than genetic disruption, suggesting this mechanism is an important driver of tumorigenesis³. Oncogenes can exhibit hypomethylation of their promoters which is associated with inappropriate expression⁴. More complicated mis-regulation of a gene can also be caused by aberrant methylation; a recent report found that hypermethylation of a p53 binding site blocked binding of the repressor, resulting in overexpression of the survivin oncogene⁵.

The identification of gene promoters that are aberrantly methylated during tumor development is valuable because it can provide insights into pathways that are commonly disrupted during tumorigenesis that can serve as drug targets^(6,7). Analysis of promoter methylation can also classify distinct subtypes of cancers that may have differential clinical characteristics in order to personalize treatment^(8,9). Finally, loci that are hypermethylated in tumors are often detected in peripheral samples (e.g. blood or stool) and may serve as diagnostic or prognostic biomarkers¹⁰.

Many techniques have been developed to detect DNA methylation including methods based on microarrays¹¹, quantitative PCR¹², mass-spectrometry¹³ and DNA sequencing¹⁴. The method that is the most direct and has the highest resolution involves treatment of genomic DNA with sodium bisulfite (which converts unmethylated cytosines to uracil, while leaving methylated cytosines intact) followed by sequencing of single molecules. Not only does this method determine the methylation state at each CpG position across a single molecule, but it also detects sequence variants. This cis information makes it possible to distinguish allele specific methylation¹⁴, and is also valuable for quantifying densely methylated molecules in a background of unmethylated or sparsely methylated molecules.

The recent introduction of second-generation DNA sequencing technologies has significantly reduced the cost required to sequence DNA. This has led to several new approaches for studying aberrant methylation using bisulfite PCR and sequencing. Methods for genome-wide surveys of methylation in a small number of samples have been developed including whole genome bisulfite sequencing¹⁵, bisulfite sequencing large fractions of restriction digested genomic DNA¹⁶, padlock probe based strategies^(17,18) and array-based hybridization capture¹⁹. In contrast, methods for the detailed study of a few loci across many samples have been described that involve amplifying each locus individually, labeling with sample-specific barcodes and performing ultra-deep bisulfite sequencing²⁰⁻²². These methods are limited to a small number of loci because the amplification of each locus separately is laborious and requires a significant amount of patient DNA per locus queried. There is still a need for a method that enables the intermediate experiment to be performed. That is, the targeted multiplexed bisulfite PCR and sequencing of an intermediate number of loci (100-1000) across a large number of samples. In cancer research this experiment is crucial since the discoveries made in genome wide profiling of a few samples need to be validated and followed-up across large numbers of patient samples.

We sought to develop a method to perform highly multiplexed bisulfite sequencing across many patient samples simultaneously. Bisulfite treatment significantly reduces the complexity of DNA sequence by converting most Cs to Ts. It also results in molecules from the same locus having different sequences depending on their methylation state. Therefore we perform the oligo hybridization and ligation based selection of the targeted loci before bisulfite treatment. The selection is highly sensitive and specific and only one pair of oligos per locus is needed, even when selecting CpG rich loci. The PCR amplification of selected loci is performed after bisulfite. Therefore the universal primers used to amplify all loci simultaneously had to be designed to exclude C's, so that they would remain unchanged through bisulfite conversion. Since the major application of this method is likely to be in clinical specimens, we optimized the method so that it didn't require large quantities of starting genomic DNA and was compatible with the DNA degradation inherit in sodium bisulfite treatment.

We designed the method to be easy to implement in any lab with standard molecular biology techniques and reagents. We also tested that it would scale up well to process many patient samples in 96-well format. We integrated sample-specific DNA barcodes into the multiplexed amplification so that many patient samples can be pooled and sequenced simultaneously on second-generation sequencing machines. Here we present a proof-of-principle experiment in which we amplified promoter regions from 94 targeted loci simultaneously and sequenced these loci across 48 samples including colon and breast tumor and adjacent normal tissue samples. In this experiment, we characterized the promoter methylation of genes that are known to be frequently mutated in cancer. We identified several novel loci that undergo frequent tumor-specific promoter methylation, and we observed allele-specific methylation patterns that occur during tumor development. We demonstrated that this method utilizes the power of next-generation sequencing to study DNA methylation at many loci across many patient samples.

Results Overview of Bisulfite Patch PCR

Bisulfite Patch PCR begins with a restriction digest of human genomic DNA to define the ends of the fragments that will be selected (FIGS. 7A & B). Targeted loci are then selected from the genomic restriction fragments by annealing patch oligos to the ends of the targeted genomic fragments. These oligos serve as a patch between the correct fragments and universal primers (U1 & U2) (FIG. 7C). The universal primers are then ligated to the genomic fragments using a thermostable ligase (FIG. 7D). Unselected genomic DNA is then degraded with exonucleases to gain additional selectively (FIG. 7E). Selected fragments are protected from degradation by a 3′ modification on the universal primer U2 (FIG. 7E). Next, the selected fragments are treated with sodium bisulfite to convert unmethylated cytosines to uracil, leaving methylated bases intact (FIG. 7F). The universal primers do not contain cytosine bases so that the sequence remains unchanged through the bisulfite conversion. The bisulfite treated selected fragments are then all amplified together simultaneously by PCR with the universal primers (U1 & U2′) (FIG. 7G). Sample-specific DNA barcodes are incorporated into the universal primers by tailing the 5′ end with a DNA sequence that is specific to each sample and the sequencing platform primers (454 sequencing primers) (FIG. 7G). The final PCR amplicons from each of the samples can be pooled together for sequencing because the first few bases of each sequencing read will identify the sample from which that sequence originated.

Highly Multiplexed Bisulfite Sequencing of CAN Gene Promoters in Colon and Breast Cancer

To test the performance of Bisulfite Patch PCR we analyzed the promoter methylation of 94 genes that are frequently mutated in breast and colon cancers (‘CAN genes’)²⁴. We designed the patch oligos to select AluI restriction digest fragments containing at least three CpG positions within 700 bp upstream of the transcription start site. We chose 42 colon CAN gene promoters, 44 breast CAN gene promoters, 4 gene promoters that were identified as both colon and breast CAN genes, and 4 controls. The four controls include an imprinted locus, a housekeeping gene promoter, and 2 neutral loci that accumulate methylation with mitotic cell division²⁵. These targeted promoter regions ranged in length from 125 by to 581 by and totaled 25.4 Kbp (SEQ ID NOs xx-xx). To determine the amount of genomic DNA required for the Bisulfite Patch PCR, we performed gel electrophoresis of the PCR products generated with different amounts of starting DNA. We observed DNA within the expected size range from reactions that started with as much as 1 microgram and as little as 20 nanograms (ng) of human genomic DNA (FIG. 8).

We performed Bisulfite Patch PCR on 250 ng of genomic DNA from each of 48 samples in parallel in a 96-well plate. The genomic DNA was isolated from a panel of 12 colon tumors, 12 matched adjacent normal colon tissues, 12 breast tumors and 12 matched adjacent normal breast tissues (TABLE F). We incorporated a 5-bp sample-specific DNA barcode in the final PCR, pooled the amplicons from all of the samples, and sequenced the pool using the Roche/454 FLX sequencer. We obtained 97,115 reads and aligned these to the in silico bisulfite treated reference sequences of our targeted loci. We successfully amplified all 94 (100%) of the targeted loci, indicating that the method is highly sensitive. Ninety percent (87,458 reads) of all reads mapped to one of the targeted promoters, demonstrating that the method is highly specific. These results demonstrate the Bisulfite Patch PCR enables highly multiplexed bisulfite sequencing.

TABLE F Tumor Lot or DNA Patient Number Tissue Normal Age Sex Pathological Diagnosis Barcode Number A811018 Breast T 34 F invasive ductal carcinoma GAGAC 1 Breast N GACAT 1 A704203 Breast T 36 F invasive ductal carcinoma GTCGT 2 Breast N CAGAT 2 A810202 Breast T 41 F invasive ductal carcinoma AGAGC 3 Breast N AGCAT 3 A811022 Breast T 46 F invasive ductal carcinoma GTGTA 4 Breast N GTCAC 4 A810219 Breast T 47 F invasive ductal carcinoma ATAGA 5 Breast N ATATC 5 A811019 Breast T 47 F invasive ductal carcinoma GACGA 6 Breast N GCAGA 6 A810210 Breast T 48 F invasive ductal carcinoma ACGAT 7 Breast N ACTAG 7 A810220 Breast T 48 F invasive ductal carcinoma ATCAG 8 Breast N ATCGC 8 A811021 Breast T 50 F invasive ductal carcinoma GCTGT 9 Breast N GTGAG 9 A810208 Breast T 55 F invasive ductal carcinoma AGCGA 10 Breast N ACAGT 10 A810213 Breast T 58 F invasive ductal carcinoma, ACTGC 11 Poorly Differentiated Breast N ACTCT 11 A811020 Breast T 77 F invasive ductal carcinoma GCACG 12 Breast N GCTAC 12 B108099 Colon T 37 M Adenocarcinoma, mucinous CTCAT 1 Colon N CTCGA 1 A811012 Colon T 40 M Adenocarcinoma, Ulcer TATAC 2 Colon N TATGT 2 B105050 Colon T 52 F Adenocarcinoma, Moderately CGTGT 3 Differentiated Colon N CTAGC 3 B105051 Colon T 56 F Adenocarcinoma, Ulcer, CTACT 4 Moderately Differentiated Colon N CTGAC 4 A709116 Colon T 57 M Adenocarcinoma, Moderately CGAGA 5 Differentiated Colon N CGCAG 5 A709121 Colon T 57 M Adenocarcinoma, Moderately CGCGC 6 Differentiated Colon N CGTAC 6 A811013 Colon T 62 F Adenocarcinoma TGCAC 7 Colon N TGCGT 7 A811015 Colon T 65 M Adenocarcinoma TGTCG 8 Colon N TCAGC 8 A811010 Colon T 71 F Adenocarcinoma, Ulcer TACAG 9 Colon N TACGC 9 A811016 Colon T 75 M Adenocarcinoma TCGAC 10 Colon N TCGTG 10 A811014 Colon T 79 M Adenocarcinoma, Ulcer TGTAT 11 Colon N TGTGA 11 A704198 Colon T 81 M Adenocarcinoma, Moderately CATAG 12 Differentiated Colon N CATGC 12

Coverage of Promoters and Reproducibility

To analyze the uniformity of the sequence coverage, we graphed the number of reads obtained for each targeted promoter versus the length of the targeted region. (FIG. 9A; TABLE G). The abundance of each promoter ranged from 10 to 5114 reads. We calculated that 93% of promoters have coverage within 10 fold of the median coverage (444 reads). There is a strong inverse correlation between amplicon length and the number of reads (linear regression R²=0.42). This correlation indicates that longer amplicons are less abundant in the reaction. If we had restricted our design to a maximum target length of 300 bp, then 92% (57/62) of those promoters would have coverage within 5 fold of the median coverage (1051 reads). These calculations indicate that approximately half of the difference in abundance of the loci is attributable to length bias. While length bias can occur in multiplex PCR, in previous versions of this universal PCR used in nested patch PCR we did not observe a correlation between amplification efficiency and length²³. Since the main difference between these methods is the sodium bisulfite treatment, we suspect that longer loci were more likely to be damaged during bisulfite conversion²⁶, and thus are less abundant in the reaction.

To test if bisulfite patch PCR reproducibly amplifies selected loci, we calculated the number of reads per locus in each of the 48 samples that were prepared in parallel. We then calculated the correlation coefficient, r, for the number of reads per locus between all possible pairs of samples. The histogram of r values obtained for the pair-wise correlations between all 48 samples is depicted in FIG. 9B. The mean r value is 0.91, indicating that the number of reads per locus is highly reproducible across patient samples. This indicates that the abundance of each locus in the reaction is not stochastic, but represents something intrinsic to the locus, including the length, as discussed above.

TABLE G Number Length of CGs CAN of per Gene # of Amplicon Ampli- Methyl- Type Gene Accession Reads (bp) con ated BT BN CT CN Breast DPYD NM_000110 1207 214 6 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast XDH NM_000379 313 276 3 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast CYP1A1 NM_000499 478 259 23 N (0%) 0/12 (0%) 0/12 (0%) 0/11 (0%) 0/12 Breast DPAGT1 NM_001382 278 182 7 N (0%) 0/12 (0%) 0/11 (0%) 0/12 (0%) 0/10 Breast CLCN3 NM_001829 2405 163 15 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast MYH9 NM_002473 167 368 31 N (0%) 0/11 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast PRPF4B NM_003913 997 225 10 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast TIMELESS NM_003920 12 308 17 N (0%) 0/4 (0%) 0/2 (0%) 0/2 (0%) 0/2 Breast LRRFIP1 NM_004735 464 165 14 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast NUP214 NM_005085 1963 201 12 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast TLN1 NM_006289 282 297 22 N (0%) 0/12 (0%) 0/11 (0%) 0/12 (0%) 0/12 Breast ABCB8 NM_007188 2390 179 8 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast ZNF646 NM_014699 2451 202 7 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast PDCD11 NM_014976 891 246 17 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast MAPKBP1 NM_014994 221 382 26 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast C14orf100 NM_016475 556 287 34 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast NOTCH1 NM_017617 81 282 34 N (0%) 0/9 (0%) 0/10 (0%) 0/12 (0%) 0/7 Breast SULF2 NM_018837 2094 211 21 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast KIAA0999 NM_025164 682 252 13 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast PLEKHA8 NM_032639 71 334 40 N (0%) 0/9 (0%) 0/10 (0%) 0/7 (0%) 0/9 Breast FLJ40869 NM_182625 385 245 31 N (0%) 0/11 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast TMEM123 NM_052932 329 383 11 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast KIAA0427 NM_014772 450 220 15 N (8%) 1/12 (8%) 1/12 (17%) 2/12 (8%) 1/12 Breast VEPH1 NM_024621 1563 210 5 N (8%) 1/12 (8%) 1/12 (8%) 1/12 (0%) 0/12 Breast SLC8A3 NM_182932 70 304 9 N (0%) 0/10 (0%) 0/8 (0%) 0/11 (0%) 0/10 Breast RGL1 NM_015149 10 581 60 N (0%) 0/2 (0%) 0/3 (0%) 0/3 (0%) 0/2 Colon ERCC6 NM_000124 1784 171 15 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Colon NF1 NM_000267 293 198 19 N (0%) 0/12 (0%) 0/11 (0%) 0/12 (0%) 0/12 Colon PTEN NM_000314 111 412 20 N (0%) 0/11 (0%) 0/9 (0%) 0/12 (0%) 0/9 Colon GALNS NM_000512 1005 242 26 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Colon GUCY1A2 NM_000855 37 313 15 N (0%) 0/6 (17%) 1/6 (0%) 0/4 (0%) 0/7 Colon UQCRC2 NM_003366 610 163 10 N (0%) 0/12 (0%) 0/11 (0%) 0/12 (0%) 0/12 Colon MCM3AP NM_003906 105 488 20 N (0%) 0/8 (0%) 0/11 (0%) 0/8 (0%) 0/11 Colon EPHB6 NM_004445 1842 172 13 N (0%) 0/11 (0%) 0/11 (0%) 0/12 (0%) 0/12 Colon KRAS NM_004985 18 415 53 N (0%) 0/3 (0%) 0/5 (0%) 0/4 (0%) 0/3 Colon ZNF262 NM_005095 359 302 21 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Colon SMAD4 NM_005359 634 217 21 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Colon SFRS6 NM_006275 402 338 35 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Colon SMTN NM_006932 145 397 34 N (0%) 0/12 (0%) 0/12 (0%) 0/11 (0%) 0/10 Colon KIAA0556 NM_015202 281 353 38 N (0%) 0/11 (0%) 0/11 (0%) 0/12 (0%) 0/12 Colon ADARB2 NM_018702 26 374 7 N (0%) 0/2 (17%) 1/6 (0%) 0/6 (0%) 0/5 Colon FBXW7 NM_033632 1097 204 24 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Colon DTNB NM_183361 2195 189 19 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Colon RET NM_020975 58 412 46 N (0%) 0/6 (0%) 0/8 (0%) 0/6 (0%) 0/8 Colon KIAA0367 NM_015225 170 235 18 N (8%) 1/12 (0%) 0/11 (8%) 1/12 (0%) 0/10 Colon SH3TC1 NM_018986 2876 157 10 N (8%) 1/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Colon TIAM1 NM_003253 354 283 40 N (0%) 0/12 (0%) 0/12 (8%) 1/12 (0%) 0/12 Colon C13orf7 NM_024546 351 269 10 N (0%) 0/11 (8%) 1/12 (17%) 2/12 (0%) 0/12 Control HSP NM_007355 155 381 21 N (0%) 0/11 (0%) 0/12 (0%) 0/10 (0%) 0/11 Dual TP53 NM_000546 1132 154 6 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Dual PIK3CA NM_006218 989 270 15 N (0%) 0/12 (0%) 0/12 (0%) 0/12 (0%) 0/12 Breast TECTA NM_005422 168 393 7 Y (100%) 11/11 (100%) 11/11 (100%) 12/12 (91%) 10/11 Breast KIAA0467 NM_015284 332 267 6 Y (100%) 12/12 (100%) 12/12 (92%) 11/12 (100%) 12/12 Breast RP1L1 NM_178857 416 221 7 Y (100%) 12/12 (100%) 12/12 (100%) 12/12 (100%) 12/12 Breast LOC340156 NM_001012418 1481 175 3 Y (100%) 12/12 (100%) 12/12 (83%) 10/12 (100%) 12/12 Breast DBN1 NM_004395 3750 165 3 Y (100%) 12/12 (100%) 12/12 (100%) 12/12 (92%) 11/12 Breast CENTG1 NM_014770 3613 190 3 Y (100%) 12/12 (100%) 12/12 (92%) 11/12 (100%) 12/12 Breast KIAA1946 NM_177454 1667 212 5 Y (100%) 12/12 (100%) 12/12 (100%) 12/12 (100%) 12/12 Breast CMYA1 NM_194293 127 173 4 Y (100%) 11/11 (89%) 8/9 (83%) 10/12 (70%) 7/10 Breast AEGP NM_206920 902 221 6 Y (83%) 10/12 (100%) 12/12 (83%) 10/12 (100%) 12/12 Breast TAF1 NM_004606 2315 153 12 Y (58%) 7/12 (67%) 8/12 (25%) 3/12 (25%) 3/12 Breast RPGRIP1 NM_020366 48 562 7 Y (88%) 7/8 (100%) 7/7 (100%) 9/9 (43%) 3/7 Breast SLC9A10 NM_183061 2096 195 3 Y (100%) 12/12 (100%) 12/12 (33%) 4/12 (75%) 9/12 Breast COL19A1 NM_001858 3069 168 4 Y (58%) 7/12 (58%) 7/12 (33%) 4/12 (8%) 1/12 Breast ABP1 NM_001091 200 207 5 Y (36%) 4/11 (67%) 8/12 (64%) 7/11 (58%) 7/12 Breast CSPP1 NM_024790 476 330 3 Y (17%) 2/12 (42%) 5/12 (25%) 3/12 (67%) 8/12 Breast NCB5OR NM_016230 282 434 6 Y (42%) 5/12 (17%) 2/12 (50%) 6/12 (18%) 2/11 Colon ITGAE NM_002208 348 349 9 Y (100%) 12/12 (100%) 12/12 (100%) 12/12 (100%) 12/12 Colon TGM3 NM_003245 178 418 5 Y (100%) 10/10 (100%) 12/12 (100%) 11/11 (100%) 10/10 Colon DSCAML1 NM_020693 2052 205 7 Y (100%) 12/12 (100%) 12/12 (100%) 12/12 (100%) 12/12 Colon TNN NM_022093 2659 161 3 Y (100%) 12/12 (100%) 12/12 (100%) 12/12 (100%) 12/12 Colon ACSL5 NM_016234 1280 235 5 Y (100%) 12/12 (100%) 12/12 (83%) 10/12 (92%) 11/12 Colon SEC8L1 NM_021807 221 345 4 Y (82%) 9/11 (100%) 10/10 (100%) 12/12 (100%) 12/12 Colon PCDHA9 NM_014005 1761 247 4 Y (83%) 10/12 (92%) 11/12 (83%) 10/12 (100%) 12/12 Colon C1QR1 NM_012072 1646 172 8 Y (100%) 12/12 (83%) 10/12 (67%) 8/12 (50%) 6/12 Colon STAB1 NM_015136 46 511 16 Y (86%) 6/7 (100%) 9/9 (70%) 7/10 (50%) 4/8 Colon HAPLN1 NM_001884 108 371 11 Y (91%) 10/11 (83%) 5/6 (30%) 3/10 (55%) 6/11 Colon BCL9 NM_004326 266 256 5 Y (67%) 8/12 (64%) 7/11 (0%) 0/12 (25%) 3/12 Colon SCN3B NM_018400 98 382 11 Y (27%) 3/11 (13%) 1/8 (0%) 0/11 (10%) 1/10 Colon GPR158 NM_020752 125 315 37 Y (8%) 1/12 (11%) 1/9 (27%) 3/11 (0%) 0/9 Colon HIST1H1B NM_005322 962 208 3 Y (25%) 3/12 (17%) 2/12 (25%) 3/12 (8%) 1/12 Colon NUP210 NM_024923 68 419 24 Y (0%) 0/8 (25%) 2/8 (11%) 1/19 (20%) 2/10 Control NKX2-5 NM_004387 1980 184 10 Y (100%) 12/12 (100%) 12/12 (100%) 12/12 (92%) 11/12 Control SOX10 NM_006941_1 1170 213 22 Y (100%) 12/12 (67%) 8/12 (100%) 12/12 (100%) 12/12 Control H19 AK311497 1614 177 9 Y (58%) 7/12 (83%) 10/12 (75%) 9/12 (92%) 11/12 Breast ICAM5 NM_003259 1717 178 11 Y (33%) 4/12 (8%) 1/12 (58%) 7/12 (0%) 0/12 Breast PPM1E NM_014906 2932 170 10 Y (25%) 3/12 (0%) 0/12 (42%) 5/12 (0%) 0/12 Colon IGFBP3 NM_000598 791 276 25 Y (67%) 8/12 (8%) 1/12 (75%) 9/12 (25%) 3/12 Colon UHRF2 NM_152896 800 185 10 Y (58%) 7/12 (8%) 1/12 (50%) 6/12 (8%) 1/12 Colon KCNQ5 NM_019842 181 273 21 Y (0%) 0/10 (0%) 0/12 (92%) 11/12 (33%) 4/12 Colon CLSTN2 NM_022131 80 304 35 Y (0%) 0/8 (10%) 1/10 (56%) 5/9 (0%) 0/10 Colon APC NM_000038 42 395 16 Y (29%) 2/7 (0%) 0/3 (0%) 0/8 (0%) 0/6 Dual LAMA1 NM_005559 438 169 14 Y (50%) 6/12 (8%) 1/12 (67%) 8/12 (17%) 2/12 Dual SORL1 NM_003105 5114 125 3 Y (33%) 4/12 (92%) 11/12 (0%) 0/12 (42%) 5/12

Allele Sensitivity at Imprinted Locus

We next sought to determine if methylated and unmethylated molecules from the same locus are amplified with similar efficiencies. This is requisite if the method is to be used to make quantitative measurements of promoter methylation. The imprinted region from the H19 locus (AK311497), which was included as a control, allows the direct comparison of the amplification efficiency of methylated and unmethylated alleles. We identified nine patients in our panel who were heterozygous for a SNP (rs2251375) in the H19 locus. We used this SNP to identify allele-specific methylation and to quantify the number of sequencing reads obtained for each allele. Allele specific methylation was observed, and both alleles were amplified with nearly equal efficiencies (FIG. 10). Imprinting methylation was observed on either allele in different individuals, consistent with the parent-of-origin determining which allele is methylated, and both alleles were represented at similar frequencies—on average 42% of the sequencing reads corresponded to the ‘G’ allele, 58% to the ‘T’ allele. Thus, our method amplifies methylated and unmethylated molecules from the same locus with nearly equal efficiency, which is crucial for quantifying heterogeneous methylation within tumors.

CAN Gene Promoter Methylation

We next examined the methylation patterns found at the targeted CAN gene promoters to determine if they exhibited tumor specific methylation. Since these genes were previously shown to be frequently mutated in colon and breast tumors²⁴, we hypothesized that the promoters of these genes might also be frequently hyper- or hypomethylated in these cancers. (TABLE H, Detailed in TABLE G).

Approximately half, (51/94), of all the promoters were unmethylated in all tissue types that we tested, including, the negative control promoter of the housekeeping gene HSP90AB1 (NM_(—)007355). Approximately one third, (34/94), of all promoters were methylated in both cancer and normal tissue including all 3 (100%) of the positive control gene promoters, the H19 imprinted promoter (AK311497) and two neutral loci that accumulated DNA methylation with mitotic division (NM_(—)006941 Exon 2, and NM_(—)004387 3′ UTR)²⁵. The remaining nine promoters exhibited tumor-specific aberrant methylation.

TABLE H Colon Breast Dual CAN CAN CAN genes genes genes Controls Total Unmethylated 22 26 2 1 51 Methylated In Tumor and 15 16 0 3 34 Normal Tissues Tumor Specific 2 2 2 0 6 Methylation: Breast & Colon Tumor Specific 2 0 0 0 2 Methylation: Colon Tumor Specific 1 0 0 0 1 Methylation: Breast Total 42 44 4 4 94

Tumor Specific Promoter Methylation

Of the nine promoters that exhibited tumor specific methylation, 5 were promoters from colon CAN genes, 2 were promoters from breast CAN genes, and 2 were promoters from genes that were frequently mutated in both colon and breast cancer (‘dual CAN genes’) (TABLE H, Detailed in TABLE G).

Five promoters exhibited tumor-specific hypermethylation in both breast and colon tumors (IGFBP3, UHRF2, LAMA1, ICAM5, PPM1E). One promoter (SORL1) exhibited tumor-specific hypomethylation in both types of cancer. The methylation patterns of ICAM5 and LAMA1 are depicted in FIG. 11 Panels A and B, respectively. Tumor specific promoter methylation of ICAM5³ and IGFBP3²⁷ was recently reported in different cohorts of breast and colon cancers. The other three loci are novel observations of aberrant tumor methylation. The frequent hypermethylation of these five loci in both types of tumors indicates that common molecular defects are shared between colon and breast cancer. The molecular defect could be an error in both types of tumors that directs methylation to these loci or it could suggest that the inactivation of these genes is a key step in tumorigenesis in both tissues.

These five loci that are hypermethylated in both breast and colon cancer are methylated in 25% to 75% of tumors (TABLE I). Loci that exhibit frequent tumor-specific methylation are often useful as clinical biomarkers. A valuable biomarker would occur frequently in patients' tumors and would be easily distinguished from normal samples. We calculated the sensitivity and specificity of these loci across our samples. The presence of aberrant methylation at two or more of these five methylated markers is found in 9 out of 12 breast tumors (75%), 11 out of 12 colon tumors (92%), 1 of 12 normal breast (8%) and 1 of 12 normal colon (8%). These strong classifiers of cancer vs. normal samples are good candidates for follow-up studies to evaluate their potential as biomarkers for stratifying disease subtypes or as diagnostic biomarkers that can be detected in peripheral specimens. The frequency of aberrant methylation at these loci approaches the significance of even the most common genetic mutations such as APC or TP53 mutations, which are reported to occur in 40-80% of tumors²⁸. This supports the previously proposed hypothesis that epigenetic defects at CAN genes may be more frequent than genetic mutations³.

Three of the CAN gene promoters show tumor specific methylation in only one type of cancer. Colon tumor specific methylation was found in the promoters of KCNQ5 (NM_(—)019842) and CLSTN2 (NM_(—)022131), and those methylation patterns are depicted in FIG. 11 Panels C and D, respectively. Breast tumor specific methylation was found in the promoter of APC (NM_(—)000038). The frequency of these aberrant events in each tumor type is cataloged in TABLE I and suggests that these loci may represent frequent tumor-specific epimutations which merit follow up investigation in a larger cohort of tumors, adjacent normal and cancer-free patient's tissue.

TABLE I Promoters Exhibiting Tumor Specific Methylation

Gene promoters exhibiting tumor specific hyper-methylation in both breast and colon tumors are not shaded. Gene promoters exhibiting tumor specific hyper-methylation in one tumor type are lightly shaded. Gene promoters exhibiting tumor specific hypo-methylation in both breast and colon tumors are heavily shaded.

Allelic Tumor Methylation

The single molecule resolution of bisulfite sequencing allows us to simultaneously assess methylation status and identify single nucleotide polymorphisms (SNPs). As seen in FIG. 12, we can distinguish whether tumor specific methylation is occurring on one allele or on both alleles in individuals that are heterozygous for the SNP (rs2854744) in IGFBP3 (NM_(—)000598). Although some aberrant promoter methylation events are known to always occur on both alleles, such as MLH1 promoter methylation²⁹, we found examples in which aberrant methylation was observed on only one allele: Breast Cancer Patient 4 acquired tumor-specific methylation primarily on the A allele, while Colon Cancer Patient 6 acquired tumor-specific methylation primarily on the C allele (FIG. 12). However, other patients acquired aberrant methylation on both alleles during tumorigenesis, such as Breast Cancer Patient 6 and Colon Cancer Patient 7 (FIG. 12). If associated with silencing, this bi-allelic methylation would indicate that both copies of the gene are inactive. Some patients exhibit different allelic methylation patterns between their tumor and adjacent normal tissue: Colon Cancer Patient 12 has methylation on their A allele across all CpGs in both the tumor and the adjacent normal tissue, but as the tumor formed the C allele acquired methylation, specifically in the region of the promoter most distal from the SNP. This suggests that the accumulation of methylation on each allele can occur in different regions of the locus and can occur at different times in tumor development. This type of allelic analysis is useful for resolving intra-tumor heterogeneity of DNA methylation, identifying heterozygous and homozygous epimutations, and understanding the accumulation of aberrant DNA methylation in different tumors.

Discussion

We have developed a method to perform highly multiplexed bisulfite sequencing of many loci across many patient samples simultaneously. This method is highly sensitive and specific and integrates sample specific DNA barcodes into the library construction so that many samples can be pooled to fully utilize the power of next-generation sequencing. Many methods are being developed to perform genome-wide profiling of DNA methylation in individual samples. Bisulfite Patch PCR provides an efficient workflow to utilize second-generation sequencing to follow up and validate aberrant methylation at many loci across large numbers of samples.

In this proof-of-principle experiment, we applied this method to characterize the promoter methylation of genes that are frequently mutated in cancer. From the 94 gene promoters that we analyzed we found that approximately 10% showed tumor specific DNA methylation in breast or colon cancer when compared to adjacent normal tissue. Our data support the previously proposed hypothesis that a relatively small set of genes that are important for tumorigenesis are disrupted in multiple ways in cancers, including frequent epigenetic defects³. We found five loci that can be used to classify tumor and normal samples with high sensitivity (9/12 breast tumors, 11/12 colon tumors) and high specificity (1/12 adjacent normal breast tissues, 1/12 adjacent normal colon tissues). In some samples we observed very low-frequency methylation of these loci in the adjacent normal tissue that may represent a field defect surrounding the tumor, or it may be a part of normal variation between individuals. Follow-up studies that include larger cohorts, cancer-free control patients and peripheral samples from patients with cancer will help determine if these new molecular defects can be useful biomarkers in the clinic. We also utilized SNPs in the sequencing data to observe allele-specific methylation patterns that provide insights into the accumulation of aberrant DNA methylation during tumor development. This method would be valuable for comparing the allelic accumulation of methylation across tumors with different stages and grades to understand the timing of aberrant methylation.

The method presented here fills a gap in the arsenal of tools for the characterization of aberrant DNA methylation. It provides the high resolution of bisulfite sequencing with the throughput of sampling many loci across many samples. This enables an experimental scale that promises to be useful in the effort to understand cancer.

Methods Design of Patch Oligonucleotides

Human promoter sequence between the transcription start site (TSS) and 700 bp upstream of the TSS was downloaded from the March 2006 assembly on the UCSC Genome Browser (www.genome.ucsc.edu) for the RefSeq genes listed in SEQ ID NOs 582-675. These sequences were then scanned for AluI restriction enzyme recognition sequences, and AluI restriction fragments that were between 125 bp and 600 bp in length and containing at least 3 CpG positions were selected. A patch oligo was then designed by sequentially including base pairs from the AluI restriction site into the fragment sequence until the Tm of the patch oligo was between 62° C. and 67° C. Any fragment whose patch oligos contained repetitive elements according to the repeat masker track on the UCSC Genome Browser (www.genome.ucsc.edu) were excluded. The patch oligos were then appended with the complement universal primer sequences to result in the appropriate patch sequence. Patch oligonucleotides were synthesized by SigmaGenosys http://www.sigmaaldrich.com/Brands/Sigma_Genosys.html). Ninety-four pairs of patch oligos were ordered in a 96-well plate. The patch oligos for two loci were duplicated on the plate so that when equimolar portions were pooled from each well these two loci were twice as concentrated in the pool. This was used to measure how the concentration of patch oligos affected amplification efficiency during protocol development. Two universal primer sequences were synthesized by IDT (www.idtdna.com), including U2, which has a 5′ phosphate and a 3 carbon spacer on the 3′ end. Oligonucleotide sequences are listed in TABLE J.

TABLE J Patch Oligonucleotide Sequences Naming: Refseq Accession Number of Locus, L (left) or R (right) side SEQ ID Sequence NO: Oligo Name (universal sequence and Alul restricition site in capitals) 385 NM_000110 L taggtgggcggggtttgAGATCACCAACTACCCACACACACC 386 NM_015149 L gcaccggcgcggAGATCACCAACTACCCACACACACC 387 NM_015284 L ttgcccacctggagagcAGATCACCAACTACCCACACACACC 388 NM_182625 L ggggagaggtctggggaaAGATCACCAACTACCCACACACACC 389 NM_000379 L attctcagagtcactgctaatagAGATCACCAACTACCCACACACACC 390 NM_177454 L gcatcaccgccatcattgcttAGATCACCAACTACCCACACACACC 391 NM_004735 L cctcaggccacgctgAGATCACCAACTACCCACACACACC 392 NM_194293 L ggggaaacagagggggagaAGATCACCAACTACCCACACACACC 393 NM_183061 L gggacagtggatttctgacaaagAGATCACCAACTACCCACACACACC 394 NM_024621 L ctttttttcgttatttgctgggaAGATCACCAACTACCCACACACACC 395 NM_001829 L cagcgtccgggagcAGATCACCAACTACCCACACACACC 396 NM_004395 L ccattctcagcccctacccAGATCACCAACTACCCACACACACC 397 NM_001012418 L tgtcaatactctcggatttacaaAGATCACCAACTACCCACACACACC 398 NM_003913 L aatgcttaaccatctcgctagacAGATCACCAACTACCCACACACACC 399 NM_001858 L ggtaattggctttttaacggttgAGATCACCAACTACCCACACACACC 400 NM_016230 L cactgggaattgtgtactgatgcAGATCACCAACTACCCACACACACC 401 NM_032639 L tctagtccctattcttgttccaaAGATCACCAACTACCCACACACACC 402 NM_001091 L gaaggacttggctgggagaaAGATCACCAACTACCCACACACACC 403 NM_007188 L ccgactggccctccaAGATCACCAACTACCCACACACACC 404 NM_024790 L gaaagtcagtgccaaaacagcaAGATCACCAACTACCCACACACACC 405 NM_178857 L ggaggcccgaaagaagcAGATCACCAACTACCCACACACACC 406 NM_005085 L ttagatgtaggttggctattggtAGATCACCAACTACCCACACACACC 407 NM_017617 L cgggcggggagcAGATCACCAACTACCCACACACACC 408 NM_006289 L gtgcccgaggcctacAGATCACCAACTACCCACACACACC 409 NM_206920 L aggactcaaccagtccagcAGATCACCAACTACCCACACACACC 410 NM_004606 L cgtaaattatacaggcattcccgAGATCACCAACTACCCACACACACC 411 NM_014976 L cctcttttcttctgtatgtccatAGATCACCAACTACCCACACACACC 412 NM_052932 L tgctcagaactctgaagtgacatAGATCACCAACTACCCACACACACC 413 NM_025164 L cttgaggccacaaatgcaggaatAGATCACCAACTACCCACACACACC 414 NM_001382 L cacaactcagttcccggaaacaaAGATCACCAACTACCCACACACACC 415 NM_005422 L ctggatttcctaattttcactacAGATCACCAACTACCCACACACACC 416 NM_003920 L gttttatttgggaggaagtaaagAGATCACCAACTACCCACACACACC 417 NM_014770 L tacgatgtaaccctttttcaggcAGATCACCAACTACCCACACACACC 418 NM_020366 L tagaactactatgtaaacttgggAGATCACCAACTACCCACACACACC 419 NM_182932 L ttgtgagagacgcttgggtgAGATCACCAACTACCCACACACACC 420 NM_016475 L ggtcctagtcccgagcgAGATCACCAACTACCCACACACACC 421 NM_014994 L ggcccgagggaccgtAGATCACCAACTACCCACACACACC 422 NM_000499 L cagagcccgggcgactAGATCACCAACTACCCACACACACC 423 NM_014699 L cgggaactttcccttccttcctAGATCACCAACTACCCACACACACC 424 NM_014906 L ctaccctcacgtggttaagagtgAGATCACCAACTACCCACACACACC 425 NM_014772 L tgtgctaatggcagatgaaaaggAGATCACCAACTACCCACACACACC 426 NM_003259 L ctggctgagatgccatgataataAGATCACCAACTACCCACACACACC 427 NM_018837 L gccgcgacccgcAGATCACCAACTACCCACACACACC 428 NM_002473 L tcggggcgcggagAGATCACCAACTACCCACACACACC 429 NM_005095 L caagtctctttgctgccagcAGATCACCAACTACCCACACACACC 430 NM_004326 L aaaggaaaaagcaaagtcccattAGATCACCAACTACCCACACACACC 431 NM_022093 L ccacacgccaacagtacaagAGATCACCAACTACCCACACACACC 432 NM_183361 L ccccgtgaactccgcaAGATCACCAACTACCCACACACACC 433 NM_024923 L ctcagccagagagccccaAGATCACCAACTACCCACACACACC 434 NM_015136 L cagcccatgctcagccAGATCACCAACTACCCACACACACC 435 NM_022131 L ctccactccgactctcggaaaAGATCACCAACTACCCACACACACC 436 NM_006218 L ttctacgagcagcaggcgAGATCACCAACTACCCACACACACC 437 NM_018986 L ccgcagccggttgatcattAGATCACCAACTACCCACACACACC 438 NM_033632 L cacgggacgaggcagaAGATCACCAACTACCCACACACACC 439 NM_001884 L acaatgatgatagtggcacataaAGATCACCAACTACCCACACACACC 440 NM_000038 L gaattaaaaatagttaccagaaaAGATCACCAACTACCCACACACACC 441 NM_014005 L cttctgtccttgattactgcaggAGATCACCAACTACCCACACACACC 442 NM_005322 L caagtaacacaggcacaggacAGATCACCAACTACCCACACACACC 443 NM_019842 L ctggcaggggctttgcAGATCACCAACTACCCACACACACC 444 NM_021807 L attgatgaagaaaagacagtataAGATCACCAACTACCCACACACACC 445 NM_000598 L cattcgtgtgtacctcgtggAGATCACCAACTACCCACACACACC 446 NM_004445 L ctaaaacagtggggctcctactcAGATCACCAACTACCCACACACACC 447 NM_015225 L ccgggggaggcactcAGATCACCAACTACCCACACACACC 448 NM_152896 L caccgcgctcaacaggaaAGATCACCAACTACCCACACACACC 449 NM_018702 L acaatgacacaaaaggaagagaaAGATCACCAACTACCCACACACACC 450 NM_020752 L gaggaaagccagtttaaagaggcAGATCACCAACTACCCACACACACC 451 NM_000314 L ggctcgtttgccctaaaaatgaaAGATCACCAACTACCCACACACACC 452 NM_016234 L caggggggccctggAGATCACCAACTACCCACACACACC 453 NM_020975 L caggaggcggggaagAGATCACCAACTACCCACACACACC 454 NM_000124 L gcgagcagggcgagaaAGATCACCAACTACCCACACACACC 455 NM_000855 L cccatcctgctggagcAGATCACCAACTACCCACACACACC 456 NM_020693 L tgtcttcacctacccacccctatAGATCACCAACTACCCACACACACC 457 NM_018400 L attagccactccctagtcctagcAGATCACCAACTACCCACACACACC 458 NM_024546 L cacgtttcaatttttttcaaaacAGATCACCAACTACCCACACACACC 459 NM_003366 L ggctacatagaatataaaaacttAGATCACCAACTACCCACACACACC 460 NM_015202 L cgcacccgggcatcAGATCACCAACTACCCACACACACC 461 NM_000512 L aggaggccttcgccgAGATCACCAACTACCCACACACACC 462 NM_002208 L cacagaacacgccgttgacAGATCACCAACTACCCACACACACC 463 NM_000267 L ctggcgctgggctcAGATCACCAACTACCCACACACACC 464 NM_005559 L gattccgagaaactatgtgcccAGATCACCAACTACCCACACACACC 465 NM_005359 L caaggagcgcgggagAGATCACCAACTACCCACACACACC 466 NM_003245 L ccacccctctcaactcacaaAGATCACCAACTACCCACACACACC 467 NM_012072 L ggggctaggaactcgaggaAGATCACCAACTACCCACACACACC 468 NM_006275 L tctttcttggagccctggcAGATCACCAACTACCCACACACACC 469 NM_003253 L agggagcccctaacaaagcAGATCACCAACTACCCACACACACC 470 NM_003906 L gggcgctgccacgaAGATCACCAACTACCCACACACACC 471 NM_006932 L ccctttctcgcgtcagtgtttaAGATCACCAACTACCCACACACACC 472 NM_004985 L CTGACCGGTCTCCACAGAGAAGATCACCAACTACCCACACACACC 473 NM_007355 L ccgaaaaagagcggaggcAGATCACCAACTACCCACACACACC 474 AK311497 L gattcccatccagttgaccgAGATCACCAACTACCCACACACACC 475 NM_004387 L CCCCCGAGAGTCAGGGAGATCACCAACTACCCACACACACC 476 NM_006941_1 L CTCCTTCTTGACCTTGCCCAGATCACCAACTACCCACACACACC 477 NM_005559 L gattccgagaaactatgtgcccAGATCACCAACTACCCACACACACC 478 NM_006218 L ttctacgagcagcaggcgAGATCACCAACTACCCACACACACC 479 NM_003105 L ACAGCAAAAACTACCCTTGATCAAGATCACCAACTACCCACACACACC 480 NM_000546 L GGTGGAAAATTCTGCAAGCCAGAGATCACCAACTACCCACACACACC 481 NM_000110 R CTACCCCACCTTCCTCATTCTCTCTaggcaggcggggc 482 NM_015149 R CTACCCCACCTTCCTCATTCTCTCTtttggccctccctctcg 483 NM_015284 R CTACCCCACCTTCCTCATTCTCTCTtaccttgtgccgggcc 484 NM_182625 R CTACCCCACCTTCCTCATTCTCTCTgcggcggtgttcatgg 485 NM_000379 R CTACCCCACCTTCCTCATTCTCTCTtcagggcatgaagagttcttgg 486 NM_177454 R CTACCCCACCTTCCTCATTCTCTCTggtagaccctcacagcgtc 487 NM_004735 R CTACCCCACCTTCCTCATTCTCTCTccacccgcagggg 488 NM_194293 R CTACCCCACCTTCCTCATTCTCTCTgcctttatcttgctggctagtg 489 NM_183061 R CTACCCCACCTTCCTCATTCTCTCTtcaggcccatcatctcttactt 490 NM_024621 R CTACCCCACCTTCCTCATTCTCTCTtcattaacacttccctctccct 491 NM_001829 R CTACCCCACCTTCCTCATTCTCTCTcacgtcagtcactcacgca 492 NM_004395 R CTACCCCACCTTCCTCATTCTCTCTtcagccccatgcttagcac 493 NM_001012418 R CTACCCCACCTTCCTCATTCTCTCTgttgccttcttagtcagatggg 494 NM_003913 R CTACCCCACCTTCCTCATTCTCTCTcttcagtcaatgctagaaatgg 495 NM_001858 R CTACCCCACCTTCCTCATTCTCTCTgggagtaatgcctttcaggttt 496 NM_016230 R CTACCCCACCTTCCTCATTCTCTCTgttccttagccttggtgctga 497 NM_032639 R CTACCCCACCTTCCTCATTCTCTCTgccggtcgcaggc 498 NM_001091 R CTACCCCACCTTCCTCATTCTCTCTgacagatggaccagggcag 499 NM_007188 R CTACCCCACCTTCCTCATTCTCTCTgtgattggaggatatgttgtca 500 NM_024790 R CTACCCCACCTTCCTCATTCTCTCTtaggaacagtgtaagagcctgg 501 NM_178857 R CTACCCCACCTTCCTCATTCTCTCTcccaccctgttccagttgt 502 NM_005085 R CTACCCCACCTTCCTCATTCTCTCTcgggctgagtagtggc 503 NM_017617 R CTACCCCACCTTCCTCATTCTCTCTgagccgcgcgtcc 504 NM_006289 R CTACCCCACCTTCCTCATTCTCTCTtggggtagaaggcggag 505 NM_206920 R CTACCCCACCTTCCTCATTCTCTCTcccacctgcccgg 506 NM_004606 R CTACCCCACCTTCCTCATTCTCTCTgctcgagtcacgtggctta 507 NM_014976 R CTACCCCACCTTCCTCATTCTCTCTagaaaaaacgaggggcgcaag 508 NM_052932 R CTACCCCACCTTCCTCATTCTCTCTcgacagatttgttgcttaaatt 509 NM_025164 R CTACCCCACCTTCCTCATTCTCTCTggcggtgggaaccttc 510 NM_001382 R CTACCCCACCTTCCTCATTCTCTCTtaaagggcccgtacctctcc 511 NM_005422 R CTACCCCACCTTCCTCATTCTCTCTtgccagagtaaacagaacacca 512 NM_003920 R CTACCCCACCTTCCTCATTCTCTCTggaccggtccccg 513 NM_014770 R CTACCCCACCTTCCTCATTCTCTCTaggtccgaggtgcaatcctaaa 514 NM_020366 R CTACCCCACCTTCCTCATTCTCTCTgtaagagatcccagaggacact 515 NM_182932 R CTACCCCACCTTCCTCATTCTCTCTccaggcagcaggcg 516 NM_016475 R CTACCCCACCTTCCTCATTCTCTCTgcgggaccgtactcgt 517 NM_014994 R CTACCCCACCTTCCTCATTCTCTCTatggtggcacgatcggc 518 NM_000499 R CTACCCCACCTTCCTCATTCTCTCTccatcctggggcgc 519 NM_014699 R CTACCCCACCTTCCTCATTCTCTCTtgagcatggcctttttgtcctc 520 NM_014906 R CTACCCCACCTTCCTCATTCTCTCTcagcccacgctgccta 521 NM_014772 R CTACCCCACCTTCCTCATTCTCTCTgccaagacagcccagtctag 522 NM_003259 R CTACCCCACCTTCCTCATTCTCTCTggcaggagtgagcgac 523 NM_018837 R CTACCCCACCTTCCTCATTCTCTCTggagggagccaaatgttcc 524 NM_002473 R CTACCCCACCTTCCTCATTCTCTCTcggctcctcgccg 525 NM_005095 R CTACCCCACCTTCCTCATTCTCTCTtctgagatcccacgggtcc 526 NM_004326 R CTACCCCACCTTCCTCATTCTCTCTagttgctgctgcactggtg 527 NM_022093 R CTACCCCACCTTCCTCATTCTCTCTcttctgacttccctcctccttc 528 NM_183361 R CTACCCCACCTTCCTCATTCTCTCTggctccatccaggcttct 529 NM_024923 R CTACCCCACCTTCCTCATTCTCTCTgagggagaaggcttgggg 530 NM_015136 R CTACCCCACCTTCCTCATTCTCTCTcacccccacaggaaccc 531 NM_022131 R CTACCCCACCTTCCTCATTCTCTCTcgccggcagcagc 532 NM_006218 R CTACCCCACCTTCCTCATTCTCTCTgaggaggggcagagcc 533 NM_018986 R CTACCCCACCTTCCTCATTCTCTCTggacggagcaggcag 534 NM_033632 R CTACCCCACCTTCCTCATTCTCTCTtggttggggccccg 535 NM_001884 R CTACCCCACCTTCCTCATTCTCTCTctgtgcccagaccttgtaaag 536 NM_000038 R CTACCCCACCTTCCTCATTCTCTCTgcttctctctccgcttccc 537 NM_014005 R CTACCCCACCTTCCTCATTCTCTCTatgcttgagattcttttcctga 538 NM_005322 R CTACCCCACCTTCCTCATTCTCTCTtttcataagaatccattgggct 539 NM_019842 R CTACCCCACCTTCCTCATTCTCTCTtcgaattctaaatccggacctg 540 NM_021807 R CTACCCCACCTTCCTCATTCTCTCTtttttcagtttccttgctttta 541 NM_000598 R CTACCCCACCTTCCTCATTCTCTCTcgagactcgcccggg 542 NM_004445 R CTACCCCACCTTCCTCATTCTCTCTcctgcctgggctcg 543 NM_015225 R CTACCCCACCTTCCTCATTCTCTCTgctgcaaccatggacagc 544 NM_152896 R CTACCCCACCTTCCTCATTCTCTCTgagggggcgggtg 545 NM_018702 R CTACCCCACCTTCCTCATTCTCTCTcgccctgctcagaaagaca 546 NM_020752 R CTACCCCACCTTCCTCATTCTCTCTgctgctgctgctgc 547 NM_000314 R CTACCCCACCTTCCTCATTCTCTCTgagatgggtgcgttgagc 548 NM_016234 R CTACCCCACCTTCCTCATTCTCTCTgcctgccttggtctctgaa 549 NM_020975 R CTACCCCACCTTCCTCATTCTCTCTcagtgcgggacgcg 550 NM_000124 R CTACCCCACCTTCCTCATTCTCTCTcaaccatagacaccgccc 551 NM_000855 R CTACCCCACCTTCCTCATTCTCTCTcgggtcggactgaggg 552 NM_020693 R CTACCCCACCTTCCTCATTCTCTCTgcccttccaacccctc 553 NM_018400 R CTACCCCACCTTCCTCATTCTCTCTctttcaggcaatgatgtcatct 554 NM_024546 R CTACCCCACCTTCCTCATTCTCTCTgcaagattcctgcgaatgtgta 555 NM_003366 R CTACCCCACCTTCCTCATTCTCTCTccgtgaaacaggggcct 556 NM_015202 R CTACCCCACCTTCCTCATTCTCTCTccacttactgagcccgc 557 NM_000512 R CTACCCCACCTTCCTCATTCTCTCTgtgtgcggatggggc 558 NM_002208 R CTACCCCACCTTCCTCATTCTCTCTtccagcccagggtcctc 559 NM_000267 R CTACCCCACCTTCCTCATTCTCTCTagagattgagagcgcggct 560 NM_005559 R CTACCCCACCTTCCTCATTCTCTCTtggcctctgggtccc 561 NM_005359 R CTACCCCACCTTCCTCATTCTCTCTttcctttctcccggctgc 562 NM_003245 R CTACCCCACCTTCCTCATTCTCTCTtggggagaagggggcag 563 NM_012072 R CTACCCCACCTTCCTCATTCTCTCTctgccgggtccctgg 564 NM_006275 R CTACCCCACCTTCCTCATTCTCTCTcgggaggcgggct 565 NM_003253 R CTACCCCACCTTCCTCATTCTCTCTccgattgggccgcc 566 NM_003906 R CTACCCCACCTTCCTCATTCTCTCTatgttctgctacaagtctaaga 567 NM_006932 R CTACCCCACCTTCCTCATTCTCTCTgcccgtccagccg 568 NM_004985 R CTACCCCACCTTCCTCATTCTCTCTATCGATGCGTTCCGCG 569 NM_007355 R CTACCCCACCTTCCTCATTCTCTCTactgcgtgccccaagtc 570 AK311497 R CTACCCCACCTTCCTCATTCTCTCTgcgggtccctggg 571 NM_004387 R CTACCCCACCTTCCTCATTCTCTCTAAGACACCAGGCTGCAGGAT 572 NM_006941_1 R CTACCCCACCTTCCTCATTCTCTCTTCCTGCGCGCTGC 573 NM_005559 R CTACCCCACCTTCCTCATTCTCTCTtggcctctgggtccc 574 NM_006218 R CTACCCCACCTTCCTCATTCTCTCTgaggaggggcagagcc 575 NM_003105 R CTACCCCACCTTCCTCATTCTCTCTCCTAGAACGCAACCAACAAGA 576 NM_000546 R CTACCCCACCTTCCTCATTCTCTCTGGACAGTCGCCATGACAA Universal Primers 577 U1 GGT GTG TGT GGG TAG TTG GTG AT 578 U2 /5Phos/AGA GAA TGA GGA AGG TGG GGT AG/3SpC3/ 579 U2′ CTA CCC CAC CTT CCT CAT TCT CT 580 454A: Sample GCC TCC CTC GCG CCA TCA G (5bp barcode) GGT GTG TGT GGG Specific Barcode: TAG TTG GTG AT U1 581 454B: Sample GCC TTG CCA GCC CGC TCA G (5bp barcode) CTA CCC CAC CTT Specific Barcode: CCT CAT TCT CT U2′

Bisulfite Patch PCR

Genomic DNA from cancer and adjacent normal tissue was obtained from Biochain (www.biochain.com) for both breast (catalog number D8235086) and colon (catalog number 8235090). Patient information and lot numbers are listed in TABLE F. Each patient sample was aliquoted into a well of a 96-well plate and digested with the AluI restriction endonuclease in 10 ul total volume reaction containing 250 ng DNA, 10 units (U) AluI enzyme (NEB), and 1× NEBuffer 2 (NEB). This reaction was incubated at 37° C. for 1 hour, followed by heat inactivation of the enzyme at 65° C. for 20 min, and held at 4° C. until the subsequent step.

Patch driven ligation of the universal primers to selected fragments was performed by addition of more reactants to the initial tube to result in the following final concentrations: 2 nM each Patch oligo, 200 nM U1 primer, 200 nM U2 primer (contains 5′ phosphate and 3′ three carbon spacer), 5 U Ampligase (Epicentre), and 1× Ampligase Reaction Buffer (Epicentre) in a total volume of 25 ul. This reaction was incubated at 95° C. for 15 minutes followed by (94° C. for 30 sec, 65° C. for 8 min) for 100 cycles, and held at 4° C.

Incorrect products, template genomic DNA and excess primer were degraded by the direct addition of 10 U Exonuclease I (USB) and 200 U Exonuclease III (Epicentre) to the reaction. This mix was incubated at 37° C. for 1 hour followed by heat inactivation at 95° C. for 20 minutes, and held at 4° C.

The reactions were then treated with sodium bisulfite to convert unmethylated cytosines to uracil. This was achieved by using the EZ DNA Methylation Gold Bisulfite Treatment Kit (Zymo Research) following the manufacturer's instructions, with one exception. Since the sample volume after the exonuclease treatment is 27 ul, the CT Conversion Reagent from the kit is made by adding 830 ul dH2O instead of 900 ul dH2O. The DNA is eluted from the columns in the final step with 10 ul M-Elution buffer.

The universal primers are then used to PCR amplify the selected bisulfite converted loci from each sample. A different pair of universal primers is used to PCR amplify each sample, and they are distinguished by a five base-pair sample-specific DNA barcode that resides between the universal primer sequence and the 454 machine specific sequence (TABLE J). There are 1,024 possible 5 bp DNA sequences, and we selected 48 sample-specific barcodes, one for each sample, that did not contain homopolymers and had the least sequence similarity to each other (The barcodes used for each patient are listed in TABLE F). For the PCR we added reagents to the last 10 ul column elution to result in these final concentrations in 50 ul: 0.5 uM each Barcoded U1, 0.5 uM each Barcoded U2′, 10 U Platinum Taq Polymerase (Invitrogen), 0.5 mM each dNTP, 2 mM MgCl₂, 0.5M Betaine, 20 mM Tris-HCl pH 8.4 and 50 mM KCl. This reaction was incubated at 93° C. for 2 minutes followed by (93° C. for 30 sec, 57° C. for 6 min) for 35 cycles, and held at 4° C. The PCR product smear between the expected sizes was confirmed by running 20 ul of the PCR product from each sample on a 3% Metaphor Agarose gel (Lonza). We then pooled 5 ul from each sample into a single tube and purified this pool on a Qiaquick Spin Column (Qiagen). The eluted DNA was quantified on the Nanodrop (www.nanodrop.com) as well as on a plate reader (BioTek Synergy HT) using PicoGreen (Invitrogen) following the manufacturer's instructions. This pooled sample was then prepared and sequenced on the 454 Life Sciences/Roche FLX machine following the manufacturer's instructions.

Sequencing Data Analysis

We obtained 97,115 sequencing reads. To determine which sequences matched our targets, we aligned the reads against a database of reference sequences for each target using WU-BLASTN (http://blast.wustl.edu). Since the sequences are sodium bisulfite treated, we substituted a T in place of C in the genomic sequence at non-CpG positions in the reference sequences. We then determined how many reads matched significantly to each promoter (BLAST smallest sum probability (P)<0.001), and put all reads from each promoter in a separate file. We computed the correlation between the number of reads and the amplicon length for each promoter using linear regression. We identified which sample each read came from by matching the first five bases of the read to the list of sample-specific barcode and corresponding patients. To determine the reproducibility of the method, we computed number of reads for each locus in each sample, and calculated the squared correlation coefficient (R²) between two samples for all possible pairs of samples. The mean of these correlation coefficients represents the average correlation between the number of reads per locus across samples. For each promoter, we used CLUSTALW to generate a multiple sequence alignment of all of the reads and the reference sequence (Larkin et al. 2007). We identified germline SNPs in the sequences by looking for variants in the reads and comparing these to known SNPs reported on the UCSC Genome Browser (www.genome.ucsc.edu). To visualize these multiple sequence alignments we create one matrix per promoter where the first column identifies the sample from which the read originated (1-48), and the remaining columns are coded for the base in the read, where C's are replaced with 8, the two alleles at SNP positions are replaced with 5 and 12, and the remaining bases are converted to 0. This matrix was then visualized as an image using the Matlab software package (The Mathworks). The matrix was sorted by sample type (the first column) and further calculations regarding the amount of methylation per read and per sample were computed using Matlab (The MathWorks Inc.).

To quantify the sensitivity and specificity of each locus exhibiting tumor-specific methylation we used a threshold to classify a locus as methylated or unmethylated in each sample. We queried many CpGs for each locus with the bisulfite sequencing. We used this information to find the optimal classifier of DNA methylation to distinguish tumor and normal samples. We search across all possible values for two parameters: % of CpGs per molecule and % of reads per sample. We found that the optimal classifier between tumor and normal was to classify a sample as ‘methylated’ if more than 20% of CpG positions per molecule were methylated in more than 35% of molecules. The fraction of samples that were classified as methylated is listed in TABLE I for each locus.

REFERENCES FOR EXAMPLE 7

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Quantitative high-throughput analysis of DNA     methylation patterns by base-specific cleavage and mass     spectrometry. Proceedings of the National Academy of Sciences of the     United States of America 102, 15785-15790 (2005). -   14. Frommer, M. et al. A genomic sequencing protocol that yields a     positive display of 5-methylcytosine residues in individual DNA     strands. Proceedings of the National Academy of Sciences of the     United States of America 89, 1827-1831 (1992). -   15. Cokus, S. J. et al. Shotgun bisulphite sequencing of the     Arabidopsis genome reveals DNA methylation patterning. Nature 452,     215-219 (2008). -   16. Meissner, A. et al. Genome-scale DNA methylation maps of     pluripotent and differentiated cells. Nature 454, 766-770 (2008). -   17. Ball, M. P. et al. Targeted and genome-scale strategies reveal     gene-body methylation signatures in human cells. Nature     biotechnology 27, 361-368 (2009). -   18. Deng, J. et al. Targeted bisulfite sequencing reveals changes in     DNA methylation associated with nuclear reprogramming. Nature     biotechnology 27, 353-360 (2009). -   19. Hodges, E. et al. High definition profiling of mammalian DNA     methylation by array capture and single molecule bisulfite     sequencing. Genome research (2009). -   20. Korshunova, Y. et al. Massively parallel bisulphite     pyrosequencing reveals the molecular complexity of breast     cancer-associated cytosine-methylation patterns obtained from tissue     and serum DNA. Genome research 18, 19-29 (2008). -   21. Taylor, K. H. et al. Ultradeep bisulfite sequencing analysis of     DNA methylation patterns in multiple gene promoters by 454     sequencing. Cancer research 67, 8511-8518 (2007). -   22. Varley, K. E., Mutch, D. G., Edmonston, T. B., Goodfellow, P. J.     & Mitra, R. D. Intra-tumor heterogeneity of MLH1 promoter     methylation revealed by deep single molecule bisulfite sequencing.     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Example 8 Nucleic Acid Patch PCR with Ends Defined by Oligonucleotide-Directed FokI Digestion

This example details creating defined ends of a nucleic acid sequence by using oligonucleotide-directed digestion on nucleic acid templates. The method is depicted in FIG. 13.

Template Preparation

FokI-directing DNA oligonucleotides were designed to anneal upstream and downstream of each of 96 targeted exons in the human genome. These loci were selected because they are genes implicated in pediatric acute lymphoblastic leukemia. The oligos contained the recognition sequence of the FokI restriction endonuclease. Human genomic DNA from the blood of healthy individuals (Promega) was incubated with FokI-directing oligonucleotides in a reaction containing appropriate buffer for the FokI enzyme, NEBuffer3 (NEB) and a final concentration of 0.1% Tween80 (Sigma) in a total volume of 9 ul. This mixture was denatured at 98° C. for 15 minutes and held at 37° C. for 5 minutes. FokI enzyme (NEB) was then added to the reaction so that there was 4 U of enzyme in a 10 ul reaction. The reaction was incubated at 37° C. for 1 hour, followed by heat inactivation of the enzyme at 65° C. for 20 min, and held at 4° C. until the subsequent step. Control reactions lacking Tween80, FokI-directing oligonucleotides, FokI enzyme, or genomic DNA were also performed.

Nucleic Acid Patch Ligation

Nucleic acid patch oligos were designed as described in Example 2 but were designed to anneal adjacent to the FokI-digested cut sites upstream and downstream of a targeted 96 exons in the human genome. Nucleic Acid Patch driven ligation of the universal primers to selected fragments was performed essentially as in Example 2. Briefly, the following reactants were added to the FokI digest to result in the following final concentrations: 2 nM each Nucleic Acid Patch oligo, 200 nM Universal Primer 1, 200 nM Universal Primer 2 with 5′ phosphate and 3′ three carbon spacer, 5 U Ampligase (Epicentre), and 1× Ampligase Reaction Buffer (Epicentre) in a total volume of 25 ul. This reaction was incubated at 95° C. for 15 minutes followed by (94° C. for 30 sec, 65° C. for 8 min) for 100 cycles, and held at 4° C.

Incorrect products, template genomic DNA and excess primer were degraded by the direct addition of 10 U Exonuclease I (USB) and 200 U Exonuclease III (Epicentre) to the reaction. This mix was incubated at 37° C. for 1 hour followed by heat inactivation at 95° C. for 20 minutes and then held at 4° C.

PCR Amplification

The universal primers were then used to PCR amplify the selected loci from each sample. For the PCR, reagents were added to the reactions to result in these final concentrations in 50 ul: 0.5 uM each Universal Primer, 10 U Platinum Taq Polymerase (Invitrogen), 0.5 mM each dNTP, 2 mM MgCl2, 0.5M Betaine, 20 mM Tris-HCl pH 8.4 and 50 mM KCl. This reaction was incubated at 93° C. for 2 minutes followed by (93° C. for 30 sec, 57° C. for 6 min) for 35 cycles, and held at 4° C. An aliquot of the reactions was analyzed by gel electrophoresis on a 2% agarose gel (Lonza).

Results

Defining template ends using oligo-directed FokI digestion was successful (FIG. 14). A smear of PCR products of the expected sizes was detected on the agarose gel. 

1. A method of amplifying at least two different nucleic acid sequences, the method comprising: a. defining the ends of at least two nucleic acid sequences, b. annealing upstream and downstream nucleic acid patches to each nucleic acid sequence of step (a), and annealing an upstream universal primer to the upstream patch, and a downstream universal primer to the downstream patch; c. ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence; and d. amplifying the nucleic acid sequences of step (c).
 2. The method of claim 1, wherein each nucleic acid sequence is encoded by genomic DNA.
 3. The method of claim 1, wherein the ends of at least two nucleic acid sequences are defined by a. annealing an upstream primer and a downstream primer to each unique nucleic acid sequence to be amplified; b. amplifying each nucleic acid sequence so as to create amplicons of the nucleic acid sequence; and c. removing the upstream and downstream primer sequences from the amplicons of step (b).
 4. The method of claim 3, wherein the upstream primer and the downstream primer of step (a) comprise uracil instead of thymine.
 5. The method of claim 3, wherein the upstream and downstream primer sequences are removed from each amplicon of step (b) in part by the addition of uracil DNA glycosylase.
 6. The method of claim 3, wherein the upstream and downstream primer sequences are removed from each amplicon of step (b) in part by the addition of an endonuclease.
 7. The method of claim 3, wherein the upstream and downstream primer sequences are removed from each unique amplicon of step (b) in part by the addition of an exonuclease.
 8. The method of claim 1, wherein the downstream universal primer comprises a protecting group.
 9. The method of claim 1, further comprising degrading non-specific amplicons after step (c) and before step (d).
 10. The method of claim 9, wherein the non-specific amplicons are degraded by contacting the amplicons with an exonuclease.
 11. The method of claim 1, further comprising sequencing the products of step (d).
 12. The method of claim 11, wherein products of step (d) are sequenced using an upstream and a downstream universal primer, wherein each primer comprises a tag specific for the unique nucleic acid sample.
 13. The method of claim 12, wherein each primer further comprises a nucleic acid sequence to prime the sequencing reaction.
 14. The method of claim 1, wherein at least 30 unique nucleic acid sequences are amplified.
 15. The method of claim 1, wherein the nucleic acid sequence is bisulfite treated DNA.
 16. The method of claim 1, wherein the ends of at least two nucleic acid sequences are defined by a method selected from the group consisting of multiplex PCR, enzyme restriction, exonuclease degradation, and triplex formation.
 17. The method of claim 16, wherein the ends of at least two nucleic acid sequences are defined by type IIS restriction enzyme digestion.
 18. A method of amplifying one or more than one unique nucleic acid sequences, the method comprising: a. annealing an upstream primer and a downstream primer to each unique nucleic acid sequence, wherein the upstream primer and the downstream primer comprise uracil instead of thymine; b. amplifying each nucleic acid sequence so as to create amplicons of the nucleic acid sequence; c. removing the upstream and downstream primer sequences from the amplicons of step (b) by contacting the amplicons with a uracil DNA glycosylase, an endonuclease, and an exonuclease; d. annealing upstream and downstream nucleic acid patches to each unique amplicon of step (c), and annealing an upstream universal primer to the upstream patch of each unique amplicon, and a downstream universal primer to the downstream patch of each unique amplicon, wherein the downstream universal primer comprises a protecting group; e. ligating the upstream universal primer and the downstream universal primer to each unique amplicon; f. degrading non-specific amplicons of step (e); and g. amplifying the amplicons of step (f).
 19. The method of claim 18, further comprising sequencing the products of step (g).
 20. The method of claim 18, wherein the non-specific amplicons are degraded by contacting the amplicons with an exonuclease. 